Method for treating a patient with neoplasia using Iressa

ABSTRACT

This invention provides a method for treating a patient with neoplasia by an adjuvant therapy that includes treatment with an inhibitor of human epidermal growth factor receptor tyrosine kinase and a cGMP-specific phosphodiesterase inhibitor.

BACKGROUND OF THE INVENTION

Virtually all of the many anti-neoplastic drugs that are currently usedin the treatment of cancer have very serious and harmful side effects.This is because cancer is generally treated with medications thatinterfere with the growth of rapidly dividing cells. Such medicationscan inhibit the growth of the cancer cells, but they almost always alsoinhibit the growth of normal cells that divide rapidly in the body. Someof the normal tissues that divide very rapidly include bone marrow(which produces blood cells), hair follicles, and intestinal epithelium.The usefulness of virtually all anti-neoplastic drugs is severelylimited by the damage they cause to these normal tissues.

This invention relates to methods for treating neoplasia using bothIressa (chemical name 4-quinazolinamine,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-(9CI))and a cyclic GMP (cGMP)-specific phosphodiesterase (PDE) inhibitor toreduce the side effects or increase the efficacy of inhibitor treatment.Iressa has been used to treat certain cancers, particularly non-smallcell lung cancer, particularly in patients where at least a first-linechemotherapy has failed. Under current practice, many types of therapyare typically used after first-line failure because they are so toxicand their side effects are so bad that the risks of the therapy outweighthe benefits until other chemotherapeutic options commonly have beenexhausted.

SUMMARY OF THE INVENTION

This invention relates to an improved method of cancer therapy thatinvolves treating a patient with both Iressa and a cyclic GMP-specificphosphodiesterase (PDE) inhibitor. The specific PDE inhibitors usefulfor this invention are compounds that inhibit both PDE5 and the types ofPDE2 described below. The novel form of PDE2 disclosed herein is fullydescribed by Liu, et al., in U.S. Pat. No. 6,200,771, A Novel CyclicGMP-Specific Phosphodiesterase And Methods For Using Same InPharmaceutical Screening For Identifying Compounds For Inhibition ofNeoplastic Lesions (for general background, see, Beavo, J. A. (1995)Cyclic nucleotide phosphodiesterases: functional implications ofmultiple isoforms. Physiological Reviews 75:725-747.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the cGMP activities of the cGMP phosphodiesterasesobtained from SW480 neoplastic cells, as assayed from the eluent from aDEAE-Trisacryl M column.

FIG. 2 is a graph of cGMP activities of the reloaded cGMPphosphodiesterases obtained from SW480 neoplastic cells, as assayed fromthe eluent from a DEAE-Trisacryl M column.

FIG. 3 is a graph of the kinetic behavior of the novel PDE of thisinvention.

FIG. 4 illustrates the effect of the sulfide derivative of sulindac andthe sulfone derivative of sulindac (a.k.a. exisulind) on purifiedcyclooxygenase activity.

FIG. 5 illustrates the effects of test compounds B and E on COXinhibition.

FIG. 6 illustrates the inhibitory effects of sulindac sulfide andexisulind on PDE4 and PDE5 purified from cultured tumor cells.

FIGS. 7A and 7B illustrate the effects of sulindac sulfide on cyclicnucleotide levels in HT-29 cells.

FIG. 8 illustrates the phosphodiesterase inhibitory activity of compoundB.

FIG. 9 illustrates the phosphodiesterase inhibitory activity of compoundE.

FIGS. 10A and 10B illustrate the effects of sulindac sulfide andexisulind on apoptosis and necrosis in HT-29 cells.

FIGS. 11A and 11B illustrate the effects of sulindac sulfide andexisulind on HT-29 cell growth inhibition and apoptosis induction asdetermined by DNA fragmentation.

FIG. 12 illustrates the apoptosis-inducing properties of compound E.

FIG. 13 illustrates the apoptosis-inducing properties of compound B.

FIG. 14 illustrates the effects of sulindac sulfide and exisulind ontumor cell growth.

FIGS. 15A and 15B illustrate the growth inhibitory andapoptosis-inducing activity of sulindac sulfide and control (DMSO).

FIG. 16 illustrates the growth inhibitory activity of compound E.

FIG. 17 illustrates the inhibition of pre-malignant, neoplastic lesionsin mouse mammary gland organ culture by sulindac metabolites.

FIG. 18A is a radiography of SDS-PAGE gel of PKG activity from SW480cells treated with drugs in the absence of added cGMP, where cells weretreated in culture for 48 hours with DMSO (0.03%, lanes 1 and 2),exisulind (200, 400 and 600 μM; lanes 3, 4, 5) and E4021 (0.1, 1 and 10μM, lanes 6, 7, 8).

FIG. 18B is a radiography of the SDS-PAGE gel of PKG activity from SW480cells treated with drugs in the presence of added cGMP, where cells weretreated in culture for 48 hours with DMSO (0.03%, lanes 1 and 2),exisulind (200, 400 and 600 μM; lanes 3, 4, 5) and E4021 (0.1, 1 and 10μM, lanes 6, 7, 8).

FIG. 19 is a bar graph of the results of Western blot experiments of theeffects of exisulind on β-catenin and PKG levels in neoplastic cellsrelative to control.

FIG. 20 is a graph of the cGMP hydrolytic activities of the cGMPphosphodiesterases obtained from HTB-26 neoplastic cells, as assayedfrom the eluent from a DEAE-Trisacryl M column.

FIG. 21 is a graph of the cGMP hydrolytic activities of the cGMPphosphodiesterases obtained from HTB-26 neoplastic cells, as assayedfrom the eluent from a DEAE-Trisacryl M column with low and highsubstrate concentration.

FIG. 22 is a graph of the cGMP hydrolytic activities of the cGMPphosphodiesterases obtained from LnCAP neoplastic cells, as assayed fromthe eluent from a DEAE-Trisacryl M column.

FIG. 23 is a graph of the cGMP hydrolytic activities of the cGMPphosphodiesterases obtained from LnCAP neoplastic cells, as assayed fromthe eluent from a DEAE-Trisacryl M column with low and high substrateconcentration.

FIG. 24 is a bar graph illustrating the specificity binding of thenon-catalytic cGMP binding sites of PDE5 for cyclic nucleotide analogsand selected PDE5 inhibitors.

FIG. 25 is a graph of the cGMP hydrolytic activities of the cGMPphosphodiesterases obtained from SW480 neoplastic cells, as assayed fromthe eluent from a DEAE-Trisacryl M column using ethylene glycol in thebuffer.

FIG. 26 is a graph of the cGMP hydrolytic activities of the cGMPphosphodiesterases obtained from SW480 neoplastic cells grown in rollerbottles, as assayed from the eluent from a DEAE-Trisacryl M column.

FIG. 27A shows a time-dependent increase in the amount ofhistone-associated fragmented DNA in LNCaP cell cultures followingtreatment with 50 μM Compound I.

FIG. 27B shows the course of treatment of PrEC prostate cells withCompound I (50 μM) that did not affect DNA fragmentation for up to 4days of treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As discussed in greater detail below, the inhibition of cGMP-specificPDEs can induce apoptosis in neoplastic cells. Human epidermal growthfactor receptor tyrosine kinase inhibitor therapies are currently usedto treat neoplasias, particularly breast cancers. The combination ofthese two types of therapies can produce an effect that neither canproduce individually, specifically achieving synergistic effects aspresented below.

As explained above, this invention among other things is a method ofcausing the use of a particular class of anti-neoplastic cGMP PDEinhibitor that acts through the pathways described herein, inconjunction with an inhibitor of human epidermal growth factor receptortyrosine kinase-based therapeutic. This method includes obtaining apharmaceutical composition that includes such an inhibitor having one ormore of the attributes set forth herein, informing physicians andpatients about those attributes, providing the pharmaceuticalcomposition to physicians and patients in need of treatment; and causinga patient to receive the pharmaceutical composition in conjunction withhuman epidermal growth factor receptor tyrosine kinase inhibitortherapy. This invention also involves obtaining a pharmaceuticalcomposition that includes a human epidermal growth factor receptortyrosine kinase inhibitor, providing the pharmaceutical composition tophysicians and patients in need of treatment; and causing a patient toreceive the pharmaceutical composition in conjunction with ananti-neoplastic cGMP PDE inhibitor.

By “informing the physician and patient,” we mean the entire range ofdirect or indirect medical educational efforts whereby pharmaceuticalsare (in effect) marketed to doctors, and thereby their patients. By wayof example only, one way is for the manufacturer to conduct the requiredmechanistic studies (of the type described below) to ascertain whetherhis compound has the attributes set forth herein, and then publishingthe results of those studies in one or more publications or includingsuch information in a package insert that accompanies thepharmaceutical. Alternatively, pharmaceutical companies can sponsor orinitiate such mechanistic studies to be performed by third parties, andthe results of those third party studies are then published.

Those reports are then used by the pharmaceutical company in its medicaleducation and marketing efforts. For example, copies of publishedreports can be distributed by the company or its agents directly todoctors at conventions or in their offices in an effort to convince thephysician that the drug indeed has the attributes that warrant use.Alternatively, those reports can be posted on the Internet directly orindirectly by the company or its agents. Also, the company can arrangefor (or have arranged for it) continuing medical education carriers toorganize events where doctors are provided such data. These and similarefforts are used by pharmaceutical companies to inform physicians andthereby the patients so that the prescribers and users of suchpharmaceuticals come to understand that the drug sold by themanufacturer has one or more of the attributes set forth herein.

By “packaged pharmaceutical,” we mean the drug (either theanti-neoplastic cGMP PDE inhibitor or the human epidermal growth factorreceptor tyrosine kinase inhibitor) as formulated in its form to beadministered to the patient) packaged in a bottle or blister card (thatmay or may not then be boxed with other bottles or blister cards), IVbag, aerosol inhaler, syringe, ointment tube, or the like. The “writtenmaterial” is that material describing said compound characterized ashaving one or more of the attributes set forth herein, and typicallycontaining directions for use in accordance with the teachings of thisinvention. One non-limiting type of written material is a packageinsert, but brochures and the like represent other types. Writtenmaterial also includes (but is not limited to) those materials inelectronic form.

The packaging can carry such written material by having the writtenmaterial affixed (releasably or otherwise) to the outside of thecontainer, or provided inside the container itself (e.g., in the case oftableted drug, an insert inside the bottle containing the tablets).Alternatively, if the bottled pharmaceutical is packaged in multiplebottles in shipping containers (e.g., boxes), one or more copies of thewritten material can be placed in the outer box. If the bottledpharmaceutical is boxed in an individual box, the written material canbe inside or on the box.

The Novel cGMP-Specific Phosphodiesterase and PDE2 from Neoplastic Cells

A. In General

One aspect of the pathway involved in this invention is the inhibitionof a PDE2 that exhibits a novel conformation and a conventional one,depending on the circumstances. In addition to inhibiting PDE5,pro-apoptotic PDE5 inhibitors inhibit this PDE2-like enzyme, whereasPDE5 inhibitors that do not induce apoptosis have not been found toinhibit this PDE2-like enzyme.

B. The Isolation of the Novel PDE Conformation

In one aspect of the pathway described herein, an isolated cGMP-specificphosphodiesterase (which appears to be a novel conformation of PDE2) wasfirst prepared from the human carcinoma cell line commonly referred toas SW480 available from the American Tissue Type Collection inRockville, Md., U.S.A. SW480 is a human colon cancer cell line thatoriginated from moderately differentiated epithelial adenocarcinoma. Asdiscussed below, a similar conformation has also been isolated fromneoplasias of the breast (i.e., HTB-26 cell line) and prostate (i.e.,LNCAP cell line).

By “isolated” we mean (as is understood in the art) not only isolatedfrom neoplastic cells, but also made by recombinant methods (e.g.,expressed in a bacterial or other non-human host vector cell lines).However, we presently believe isolation from the human neoplastic cellline is preferable since we believe that the target protein so isolatedhas a structure (i.e., a conformation or topography) that is closer to,if not identical with, one of the native conformations in the neoplasticcell. This conformation assists in the selection of anti-neoplasticcompounds that will inhibit the target enzyme(s) in vivo.

The novel PDE activity was first found in SW480 colon cancer cell lines.To isolate the novel phosphodiesterase from SW480, approximately fourhundred million SW480 cells were grown to confluence in and were scrapedfrom 150 cm² tissue culture dishes after two washes with 10 mL cold PBSand pelleted by centrifugation. The cells were re-suspended inhomogenization buffer (20 mL TMPI-EDTA-Triton pH 7.4:20 mM Tris-HOAc, 5mM MgAc₂, 0.1 mM EDTA, 0.8% Triton-100, 10 μM benzamidine, 10 μM TLCK,2000 U/mL aprotinin, 2 μM leupeptin, 2 μM pepstatin A) and homogenizedon an ice bath using a polytron tissumizer (three times, 20seconds/pulse). The homogenized material was centrifuged at 105,000 gfor 60 minutes at 4° C., in a Beckman L8 ultracentrifuge, and thesupernatant was diluted with TMPI-EDTA (60 mL) and applied to a10-milliliter DEAE-Trisacryl M column pre-equilibrated with TMPI-EDTAbuffer. The loaded column was washed with 60 mL of TM-EDTA, and PDEactivities were eluted with a 120 mL linear gradient of NaOAC (0-0.5 M)in TM-EDTA, at a flow rate of 0.95 mL/minute, 1.4 mL/fraction. Eightyfractions were collected and assayed for cGMP hydrolysis immediately(i.e. within minutes). FIG. 1. shows the column's elution profile,revealing two initial peaks of cGMP PDE activity, Peaks A and B, whichwere eluted by 40-50 mM and 70-80 mM NaOAC, respectively. As explainedbelow, Peak A is PDE5, whereas Peak B is a novel cGMP-specificphosphodiesterase activity.

Cyclic nucleotide PDE activity of each fraction was determined using themodified two-step radio-isotopic method of Thompson et al. (Thompson W.J., et al., Adv. Cyclic Nucleotide Res. 10: 69-92, 1979), as furtherdescribed below. The reaction was in 400 μl containing Tris-HCl (40 mM;pH 8.0), MgCl₂ (5 mM), 2-mercaptoethanol (4 mM), bovine serum albumin(30 μg), cGMP (0.25 μM-5 μM) with constant tritiated substrate (200,000cpm). The incubation time was adjusted to give less than 15% hydrolysis.The mixture was incubated at 30° C. followed by boiling for 45 secondsto stop the reaction. Then, the mixture was cooled, snake venom (50 μg)added, and the mixture was incubated at 30° C. for 10 minutes. MeOH (1mL) was added to stop the reaction, and the mixture was transferred toan anion-exchange column (Dowex 1-X8, 0.25 mL resin). The eluent wascombined with a second mL of MeOH, applied to the resin, and afteradding 6 mL scintillation fluid, tritium activity was measured using aBeckman LS 6500 for one minute.

To fractionate the cGMP hydrolytic activities of Peaks A and B further,fractions 15 to 30 of the original 80 were reloaded onto theDEAE-Trisacryl M column and eluted with a linear gradient of NaOAC(0-0.5 M) in TM-EDTA. Fractions were again immediately assayed for cGMPhydrolysis (using the procedure described above with 0.2, 2, 5 μMsubstrate), the results of which are graphically presented in FIG. 2.One observation about Peak B illustrated in FIG. 2 is that increasingsubstrate concentration of cGMP dramatically enhanced activity whencontrasted to Peak A. While this observation is consistent with itsbeing a PDE2, the fact that the enzyme characterized in FIG. 2 iscGMP-specific (see below) suggests that it has a novel conformationcompared to the classic PDE2 reported in the literature. Peak A activityshows apparent substrate saturation of high affinity catalytic sites.

C. The Isolation of Classic PDE2 from SW480

Two methods were found that allowed “Peak B” to be isolated from SW480so that the enzyme had the classical PDE2 activity (i.e. was notcGMP-specific, but was cGMP stimulated). The first method involvedgrowing the SW480 in 850 cm² Corning roller bottles instead of 150 cm²tissue culture flasks. SW480 were grown in roller bottles at 0.5 rpmwith each bottle containing 200 mL of RPMI 1640, 2 mM glutamine, and 25mM HEPES. Cells were harvested by the following procedure.

PBS media was warmed to 37° C. for at least 15 minutes. 200 mL of 5%FBS/RPMI 1640 complete media was prepared and 5 mL of glutamine wasadded. 5 mL of antibiotic/antimycotic was also added.

70 mL of the PBS solution was added to 10 mL of 4× Pancreatin. Themixture was maintained at room temperature. The media was removed andthe flask was rinsed with 4 mL of PBS being sure the bottom of the flaskwas covered. All solution was removed with a pipet. 4 mL of dilutedPancreatin was added to the flask, and the flask was swished to coverits bottom. The flask was incubated at 37° C. for 8-10 minutes. Afterthe incubation, the flask was quickly checked under an invertedmicroscope to make sure all cells were rounded. The flask was hitcarefully on its side several times to help detach cells. 10 mL of coldcomplete media was added to the flask to stop the Pancreatinproteolysis. The solution was swirled over the bottom to collect thecells. The media was removed using a 25 mL pipet, and the cells placedin 50 mL centrifuge tubes on ice. The tubes were spun at 1000 rpm at 4°C. for 5 minutes in a clinical centrifuge to pellet cells. Thesupernatant was poured off and each pellet frozen on liquid nitrogen for15 seconds. The harvested cells can be stored in a −70° C. freezer.

The PDEs from the harvested SW480 cells were isolated using an FPLCprocedure. A Pharmacia AKTA FPLC was used to control sample loading andelution on an 18 mL DEAE TrisAcryl M column. About 600 million cells ofSW480 were used for the profiles. After re-suspending cells inhomogenization buffer (20 mL TMPI-EDTA-Triton pH 7.4:20 mM Tris-HOAc, 5mM MgAc₂, 0.1 mM EDTA, 0.8% Triton-100, 10 μM benzamidine, 10 μM TLCK,2000 U/mL aprotinin, 2 μM leupeptin, 2 μM pepstatin A), samples weremanually homogenized. FPLC buffer A was 8 mM TRIS-acetate, 5 mM Mgacetate, 0.1 mM EDTA, pH 7.5 and buffer B was 8 mM TRIS-acetate, 5 mM Mgacetate, 0.1 mM EDTA, 1 M Na acetate, pH 7.5. Supernatants were loadedonto the column at 1 mL per minute, followed by a wash with 60 mL bufferA at 1 mL per minute. A gradient was run from 0-15% buffer B in 60 mL,15-50% buffer B in 60 mL, and 50-100% buffer B in 16 mL. During thegradient, 1.5 mL fractions were collected.

The profile obtained was similar (FIG. 26) to the profile for the novelPDE activity (see, e.g., FIG. 1) obtained above, except that Peak Bisolated in this manner showed cAMP hydrolytic activity at 0.25 μMsubstrate that could be activated 2-3 fold by 5 μM cGMP.

A second method used to isolate classic PDE2 from SW480 was done using anon-FPLC DEAE column procedure described above (see Section IIB) withthe modification that the buffers contained 30% ethylene glycol, 10 mMTLCK and 3.6 mM β-mercaptoethanol. The addition of these reagents to thebuffers causes a shift in the elution profile (see FIG. 25) from low tohigh sodium acetate so that Peak A moves from 40 to 150 mM, Peak B from75 to 280 mM and Peak C from 200 to 500 mM Na acetate (see FIG. 25).Peak B in FIG. 25 was assayed with 2 μM cAMP substrate and showed atwo-fold activation by 5 μM cGMP (see FIG. 26). The selective PDE2inhibitor EHNA inhibited 2 μM cGMP PDE activity in this Peak B with anIC₅₀ of 1.6 μM and inhibited 2.0 μM cAMP PDE activity in Peak B with anIC₅₀ of 3.8 μM (and IC₅₀ of 2.5 μM with addition of 10 μM rolipram).

D. cGMP-Specificity of PDE Peak A and the Novel Peak B Activity

Each fraction from the DEAE column from Section IIB was also assayed forcGMP-hydrolysis activity (0.25 μM cGMP) in the presence or absence ofCa⁺⁺, or Ca⁺⁺-CaM and/or EGTA and for cAMP (0.25 μM cAMP) hydrolysisactivity in the presence or absence of 5 μM cGMP. Neither PDE Peak A andPeak B (fractions 5-22; see FIG. 1) hydrolyzed cAMP significantly,establishing that neither had the activity of a classic cAMP-hydrolyzingfamily of PDE (i.e. a PDE 1, 2, 3).

Ca⁺⁺ (with or without calmodulin) failed to activate either cAMP or cGMPhydrolysis activity of either Peak A or B, and cGMP failed to activateor inhibit cAMP hydrolysis. Such results establish that Peaks A and Bconstitute cGMP-specific PDE activities but not classic or previouslyknown PDE1, PDE2, PDE3 or PDE4 activities.

For the novel PDE Peak B, as discussed below, cyclic GMP activated thecGMP hydrolytic activity of the enzyme, but did not activate any cAMPhydrolytic activity (in contrast with the Peak B from Section IICabove). This reveals that the novel PDE Peak B—the novelphosphodiesterase of this invention—is not a cGMP-stimulated cAMPhydrolysis (“cGS”) or among the classic or previously known PDE2 familyactivities because the known isoforms of PDE2 hydrolyze both cGMP andcAMP.

E. Peak A is a Classic PDE5, but the Novel Peak B—a New cGMP-SpecificPDE—is Not

To characterize any PDE isoform, kinetic behavior and substratepreference should be assessed.

Peak A showed typical “PDE5” characteristics. For example, the K_(m) ofthe enzyme for cGMP was 1.07 μM, and Vmax was 0.16 nmol/min/mg. Inaddition, as discussed below, zaprinast (IC₅₀=1.37 μM) and E4021 (IC₅₀=3nM) and sildenafil inhibited activity of Peak A. Further, zaprinastshowed inhibition for cGMP hydrolysis activity of Peak A, consistentwith results reported in the literature.

PDE Peak B from Section IIB showed considerably different kineticproperties as compared to PDE Peak A. For example, in Eadie-Hofsteeplots of Peak A, cyclic GMP hydrolysis shows single line with negativeslope with increasing substrate concentrations, indicative ofMichaelis-Menten kinetic behavior. Peak B, however, shows the novelproperty for cGMP hydrolysis in the absence of cAMP of a decreasing(apparent K_(m)=8.4), then increasing slope (K_(m)<1) of Eadie-Hofsteeplots with increasing cGMP substrate (see, FIG. 3). Thus, thisestablishes Peak B's submicromolar affinity for cGMP (i.e., whereK_(m)<1).

Consistent with the kinetic studies (i.e., FIG. 3) andpositive-cooperative kinetic behavior in the presence of cGMP substrate,was the increased cGMP hydrolytic activity in the presence of increasingconcentrations of cGMP substrate. This was discovered by comparing 0.25μM, 2 μM and 5 μM concentrations of cGMP in the presence of PDE Peak Bafter a second DEAE separation to rule out cAMP hydrolysis and to ruleout this new enzyme being a previously identified PDE5. Higher cGMPconcentrations evoked disproportionately greater cGMP hydrolysis withPDE Peak B, as shown in FIG. 2.

These observations suggest that cGMP binding to the Peak B enzyme causesa conformational change in the enzyme. This confirms the advantage ofusing the native enzyme from neoplastic cells, but this invention is notlimited to the native form of the enzyme having the characteristics setforth above.

F. Zaprinast- and Sildenafil-Insensitivity of PDE Peak B Relative toPeak A, and Their Effects on Other PDE Inhibitors

Different PDE inhibitors were studied using twelve concentrations ofdrug from 0.01 to 100 μM and substrate concentration of 0.25 μM ³H-cGMP.IC₅₀ values were calculated with variable slope, sigmoidal curve fitsusing Prism 2.01 (GraphPad). The results are shown in Table 1. Whilecompounds E4021 and zaprinast inhibited Peak A (with high affinities),IC₅₀ values calculated against the novel PDE activity in Peak B (SectionIIB) are significantly increased (>50 fold). This confirms that Peak Ais a PDE5. These data further illustrate that the novel PDE activity ofthis invention is, for all practical purposes, zaprinast-insensitive andE4021-insensitive.

TABLE 1 Comparison of PDE Inhibitors Against Peak A and Section IA PeakB (cGMP Hydrolysis) IC₅₀ IC₅₀ PDE Family Peak A Peak B Ratio (IC₅₀Compound Inhibitor (μM) (μM) Peak A/Peak B) E4021 5 0.003 8.4 0.0004Zaprinast 5 1.4 >30 <0.05 Compound E 5 and others 0.38 0.37 1.0 Sulindacsulfide 5 and others 50 50 1.0 Vinpocetine 1 >100 >100 EHNA 2 >100 3.7Indolidan 3 31 >100 <0.31 Rolipram 4 >100 >100 Sildenafil 5 .0003 >10<.00003

By contrast, sulindac sulfide and Compound E competitively inhibitedboth Peaks A and B phosphodiesterases at the same potency (IC₅₀=0.38 μMfor PDE Peak A; 0.37 μM for PDE Peak B).

There is significance for the treatment of neoplasia and the selectionof useful compounds for such treatment in the fact that Peak B (eitherform of it) is zaprinast-insensitive whereas Peaks A and B are bothsensitive to sulindac sulfide and Compound E. We have tested zaprinast,E4021 and sildenafil to ascertain whether they induce apoptosis orinhibit the growth of neoplastic cells, and have done the same forCompound E. As explained below, zaprinast by itself does not havesignificant apoptosis-inducing or growth-inhibiting properties, whereassulindac sulfide and Compound E are precisely the opposite. In otherwords, the ability of a compound to inhibit both PDE Peaks A and Bcorrelates with its ability to induce apoptosis in neoplastic cells,whereas if a compound (e.g., zaprinast) has specificity for PDE Peak Aonly, that compound will not by itself induce apoptosis.

G. Insensitivity of the Novel PDE Peak B to Incubation withcGMP-Dependent Protein Kinase G

Further differences between PDE Peak A and the novel Peak B (SectionIIB) were observed in their respective cGMP-hydrolytic activities in thepresence of varying concentrations of cGMP-dependent protein kinase G(which phosphorylates typical PDE5). Specifically, Peak A and Peak Bfractions from Section IIB were incubated with different concentrationsof protein kinase G at 30° C. for 30 minutes. Cyclic GMP hydrolysis ofboth peaks has assayed after phosphorylation was attempted. Consistentwith previously published information about PDE5, Peak A showedincreasing cGMP hydrolysis activity in response to protein kinase Gincubation, indicating that Peak A was phosphorylated. Peak B wasunchanged, however (i.e., was not phosphorylated and insensitive toincubation with cGMP-dependent protein kinase G). These data areconsistent with Peak A being an isoform consistent with the known PDE5family and Peak B from Section IIB being a novel cGMP-specific PDEactivity.

H. Novel Peak B in Prostate and Breast Cancer Cell Lines

The novel Peak B was also isolated from two other neoplastic cell lines,a breast cancer cell line, HTB-26, and a prostate cancer cell line,LnCAP, by a procedure similar to the one above used to isolate it fromSW480. The protocol was modified in several respects. To provide evengreater reproducibility to allow comparison of different cell lines, aPharmacia AKTA FPLC was used to control sample loading and elution on an18 mL DEAE TrisAcryl M column. SW840 was run by this same proceduremultiple times to provide a reference of Peak B. 200-400 million cellsof SW480 were used for the profiles. 70 million cells of LnCAP were usedfor a profile (see FIGS. 22 and 23), and in a separate experiment 32million cells of HTB-26 were used for a profile (see FIGS. 20 and 21).After re-suspending cells in homogenization buffer, samples weremanually homogenized. FPLC buffer A was 8 mM TRIS-acetate, 5 mM Mgacetate, 0.1 mM EDTA, pH 7.5 and buffer B was 8 mM TRIS-acetate, 5 mM Mgacetate, 0.1 mM EDTA, 1 M Na acetate, pH 7.5. Supernatants were loadedonto the column at 1 mL per minute, followed by a wash with 60 mL bufferA at 1 mL per minute. A gradient was run from 0-15% buffer B in 60 mL,15-50% buffer B in 60 mL, and 50-100% buffer B in 16 mL. During thegradient 1.5 mL fractions were collected. Peaks of cGMP PDE activityeluted around fraction 65 that was at 400 mM Na acetate (see FIGS.20-23). This activity was measured at 0.25 μM cGMP (indicatingsubmicromolar affinity for cGMP). Rolipram, a PDE4-specific drug,inhibited most of the cAMP PDE activity (i.e. the cAMP activity was dueto PDE4), indicating that the Peak B's cGMP activity was specific forcGMP over cAMP. None of the three Peak B's (from SW480, HTB-26, andLnCAP) showed stimulation with calcium/calmodulin and all were resistantto 100 nM E4021, a specific PDE5-specific inhibitor like zaprinast (seeFIGS. 20 and 22). The Peak B's also showed a dramatic increase inactivity when substrate was increased from 0.25 μM to 5 μM cGMP(suggesting positively cooperative kinetics) (see FIGS. 21 and 23).Also, the three peaks show similar inhibition by exisulind and CompoundI, below.

Protein Kinase G and β-Catenin Involvement—in General

A series of experiments were performed to ascertain what effect, if any,an anti-neoplastic cGMP-specific PDE inhibitor such as exisulind had oncGMP-dependent protein kinase G (“PKG”) in neoplastic cells containingeither the adenomatous polyposis coli gene (“APC gene”) defect or adefect in the gene coding for β-catenin. As explained below, such aninhibitor causes an elevation in PKG activity in such neoplastic cells.That increase in activity was not only due to increased activation ofPKG in cells containing either defect, but also to increased expressionof PKG in cells containing the APC defect. In addition, when PKG fromneoplastic cells with either defect is immunoprecipitated, itprecipitates with β-catenin.

β-catenin has been implicated in a variety of different cancers becauseresearchers have found high levels of it in patients with neoplasiascontaining mutations in the APC tumor-suppressing gene. People withmutations in this gene at birth often develop thousands of small tumorsin the lining of their colon. When it functions properly, the APC genecodes for a normal APC protein that is believed to bind to and regulateβ-catenin. Thus, the discovery that PKG in neoplastic cells containingeither the APC gene defect or the β-catenin defect is bound to β-cateninindeed strongly implicates PKG in one of the major cellular pathwaysthat leads to cancer. In addition, the relationship betweencGMP-specific inhibition and PKG elevation upon treatment with SAANDslinks cGMP to the PKG/β-catenin/APC defect in such cells.

This latter link is further buttressed by the observation that β-cateninitself is reduced when neoplastic cells containing the APC defect or theβ-catenin defect are exposed to a SAAND. This reduction in β-catenin isinitiated by PKG itself. PKG phosphorylates β-catenin—which is anothernovel observation associated with this invention. The phosphorylation ofβ-catenin allows β-catenin to be degraded by ubiquitin-proteasomalsystem.

This phosphorylation of β-catenin by PKG is important in neoplasticcells because it circumvents the effect of the APC and β-cateninmutations. The mutated APC protein affects the binding of the β-cateninbound to the mutant APC protein, which change in binding has heretoforebeen thought to prevent the phosphorylation of β-catenin by GSK-3bkinase. In the case of mutant β-catenin, an elevation of PKG activityalso allows the mutant β-catenin to be phosphorylated. Elevating PKGactivity in neoplasia with cGMP-PDE inhibition allows for β-cateninphosphorylation (leading to its degradation) in neoplastic cellscontaining either type of mutation.

In short, these findings not only lead to new pharmaceutical screeningmethods to identify further SAAND candidate compounds, but also buttressthe role of cGMP-specific PDE inhibition in therapeutic approaches toneoplasia. This observation may also explain the unexpectedly broadrange of neoplasias SAANDs can inhibit since both neoplasia with andwithout the APC defect can be treated, as explained above.

IV. Screening Pharmaceutical Compositions using the PDES

A. In General

The novel PDE of this invention and PDE2 are useful with or without PDE5to identify compounds that can be used to treat or prevent neoplasias,and that are not characterized by serious side effects.

Cancer and precancer may be thought of as diseases that involveunregulated cell growth. Cell growth involves a number of differentfactors. One factor is how rapidly cells proliferate, and anotherinvolves how rapidly cells die. Cells can die either by necrosis orapoptosis depending on the type of environmental stimuli. Celldifferentiation is yet another factor that influences tumor growthkinetics. Resolving which of the many aspects of cell growth is affectedby a compound is important to the discovery of a relevant target forpharmaceutical therapy. Screening assays based on this technology can becombined with other tests to select compounds that have growthinhibiting and pro-apoptotic activity.

This invention is the product of several important discoveries. First,the present inventors discovered that desirable inhibitors of tumor cellgrowth induce premature death of cancer cells by apoptosis (see, Piazza,G. A., et al., Cancer Research, 55(14), 3110-16, 1995). Second, severalof the present inventors unexpectedly discovered compounds thatselectively induce apoptosis without substantial COX inhibition alsoinhibit PDE5. In particular, and contrary to leading scientific studies,desirable compounds for treating neoplastic lesions inhibit PDE5 (EC3.1.4.17). PDE5 is one of at least ten gene families ofphosphodiesterase. PDE5 and the novel PDE of this invention are uniquein that they selectively degrade cyclic GMP and not cAMP, while theother families of PDE selectively degrade/hydrolyze cAMP and not cGMP ornon-selectively degrade both cGMP and cAMP. Preferably, desirablecompounds used to treat neoplasia do not substantially inhibitnon-selective or cAMP degrading phosphodiesterase types.

B. COX Screening

A preferred embodiment of the present invention involves determining thecyclooxygenase inhibition activity of a given compound, and determiningthe cGMP specific PDE inhibitory activity of the compound. The testcompounds are assessed for their ability to treat neoplastic lesionseither directly or indirectly by comparing their activities againstknown compounds useful for treating neoplastic lesions. A standardcompound that is known to be effective for treating neoplastic lesionswithout causing gastric irritation is5-fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenylacetic acid(“exisulind”). Other useful compounds for comparative purposes includethose that are known to inhibit COX, such as indomethacin and thesulfide metabolite of sulindac:5-fluoro-2-methyl-1-(p-methylsulfinylbenzylidene)-3-indenylacetic acid(“sulindac sulfide”). Other useful compounds for comparative purposesinclude those that are known to inhibit cGMP-specific PDEs, such as1-(3-chloroanilino)-4-phenyphthalazine (“MY5445”).

As used herein, the term “precancerous lesion” includes syndromesrepresented by abnormal neoplastic, including dysplastic, changes oftissue. Examples include dysplastic growths in colonic, breast, prostateor lung tissues, or conditions such as dysplastic nevus syndrome, aprecursor to malignant melanoma of the skin. Examples also include, inaddition to dysplastic nevus syndromes, polyposis syndromes, colonicpolyps, precancerous lesions of the cervix (i.e., cervical dysplasia),esophagus, lung, prostatic dysplasia, prostatic intraneoplasia, breastand/or skin and related conditions (e.g., actinic keratosis), whetherthe lesions are clinically identifiable or not.

As used herein, the terms “carcinoma” or “cancer” refers to lesionswhich are cancerous. Examples include malignant melanomas, breastcancer, prostate cancer and colon cancer. As used herein, the terms“neoplasia” and “neoplasms” refer to both cancerous and pre-cancerouslesions.

As used herein, the abbreviation PG represents prostaglandin; PSrepresents prostaglandin synthetase; PGE₂ represents prostaglandin E₂;PDE represents phosphodiesterase; COX represents cyclooxygenase; cyclicnucleotide, RIA represents—radioimmunoassay.

COX inhibition by a compound can be determined by either of two methods.One method involves measuring PGE₂ secretion by intact HL-60 cellsfollowing exposure to the compound being screened. The other methodinvolves measuring the activity of purified cyclooxygenases (COXs) inthe presence of the compound. Both methods involve protocols previouslydescribed in the literature, but preferred protocols are set forthbelow.

Compounds can be evaluated to determine whether they inhibit theproduction of prostaglandin E₂ (“PGE₂”), by measuring PGE₂. Using anenzyme immunoassay (EIA) kit for PGE₂, such as commercially availablefrom Amersham, Arlington Heights, Ill. U.S.A. Suitable cells includethose that make an abundance of PG, such as HL-60 cells. HL-60 cells arehuman promyelocytes that are differentiated with DMSO into maturegranulocytes (see, Collins, S. J., Ruscetti, F. W., Gallagher, R. E. andGallo, R. C., “Normal Functional Characteristics of Cultured HumanPromyelocytic Leukemia Cells (HL-60) After Induction of DifferentiationBy Dimethylsulfoxide”, J. Exp. Med., 149:969-974, 1979). Thesedifferentiated cells produce PGE₂ after stimulation with a calciumionophore, A23187 (see, Kargman, S., Prasit, P. and Evans, J. F.,“Translocation of HL-60 Cell 5-Lipoxygenase”, J. Biol. Chem., 266:23745-23752, 1991). HL-60 cells are available from the ATCC(ATCC:CCL240). They can be grown in a RPMI 1640 medium supplemented with20% heat-inactivated fetal bovine serum, 50 U/mL penicillin and 50 μg/mLstreptomycin in an atmosphere of 5% CO₂ at 37° C. To induce myeloiddifferentiation, cells are exposed to 1.3% DMSO for 9 days and thenwashed and resuspended in Dulbecco's phosphate-buffered saline at aconcentration of 3×10⁶ cells/mL.

The differentiated HL-60 cells (3×10⁶ cells/mL) are incubated for 15minutes at 37° C. in the presence of the compounds tested at the desiredconcentration. Cells are then stimulated by A23187 (5×10⁻⁶ M) for 15minutes. PGE₂ secreted into the external medium is measured as describedabove.

As indicated above, a second method to assess COX inhibition of acompound is to measure the COX activity in the presence of a testcompound. Two different forms of cyclooxygenase (COX-I and COX-2) havebeen reported in the literature to regulate prostaglandin synthesis.COX-2 represents the inducible form of COX while COX-I represents aconstitutive form. COX-I activity can be measured using the methoddescribed by Mitchell et al. (“Selectivity of NonsteroidalAnti-inflammatory Drugs as Inhibitors of Constitutive and InducibleCyclooxygenase,” Proc. Natl. Acad. Sci. USA., 90:11693-11697, 1993,which is incorporated herein by reference) using COX-I purified from ramseminal vesicles as described by Boopathy & Balasubramanian,“Purification And Characterization Of Sheep Platelet Cyclooxygenase”(Biochem. J., 239:371-377, 1988, which is incorporated herein byreference). COX-2 activity can be measured using COX-2 purified fromsheep placenta as described by Mitchell et al., 1993, supra.

The cyclooxygenase inhibitory activity of a drug can be determined bymethods known in the art. For example, Boopathy & Balasubramanian, 1988,supra, described a procedure in which prostaglandin H synthase 1 (CaymanChemical, Ann Arbor, Mich.) is incubated at 37° C. for 20 minutes with100 μM arachidonic acid (Sigma Chemical Co.), cofactors (such as 1.0 mMglutathione, 1.0 mM hydroquinone, 0.625 μM hemoglobin and 1.25 mM CaCl₂in 100 mM Tris-HCl, pH 7.4) and the drug to be tested. Followingincubation, the reaction can be terminated with trichloroacetic acid.After stopping the reaction by adding thiobarbituric acid andmalonaldehyde, enzymatic activity can then be measuredspectrophotometrically at 530 nm.

Obviously, a compound that exhibits a lower COX-I or COX-2 inhibitoryactivity in relation to its greater combined PDE5/novel PDE/PDE2inhibitory activities may be a desirable compound.

The amount of COX inhibition is determined by comparing the activity ofthe cyclooxygenase in the presence and absence of the test compound.Residual (i.e., less than about 25%) or no COX inhibitory activity at aconcentration of about 100 μM is indicative that the compound should beevaluated further for usefulness for treating neoplasia.

C. Determining Phosphodiesterase Inhibition Activity

Compounds can be screened for inhibitory effect on the activity of thenovel phosphodiesterase of this invention using either the enzymeisolated as described above, a recombinant version, or using the novelPDE and/or PDE2 together with PDE5. Alternatively, cyclic nucleotidelevels in whole cells are measured by RIA and compared to untreated andzaprinast-treated cells.

Phosphodiesterase activity can be determined using methods known in theart, such as a method using radioactive ³H cyclic GMP (cGMP)(cyclic3′,5′-guanosine monophosphate) as the substrate for the PDE enzyme.(Thompson, W. J., Teraski, W. L., Epstein, P. M., Strada, S. J.,Advances in Cyclic Nucleotide Research, 10:69-92, 1979, which isincorporated herein by reference). In brief, a solution of definedsubstrate ³H-cGMP specific activity (0.2 μM; 100,000 cpm; containing 40mM Tris-HCl (pH 8.0), 5 mM MgCl₂ and 1 mg/mL BSA) is mixed with the drugto be tested in a total volume of 400 μl. The mixture is incubated at30° C. for 10 minutes with isolated PDE of this invention. Reactions areterminated, for example, by boiling the reaction mixture for 75 seconds.After cooling on ice, 100 μl of 0.5 mg/mL snake venom (O. Hannah venomavailable from Sigma) is added and incubated for 10 minutes at 30° C.This reaction is then terminated by the addition of an alcohol, e.g. 1mL of 100% methanol. Assay samples are applied to 1 mL Dowex 1-X8column; and washed with 1 mL of 100% methanol. The amount ofradioactivity in the breakthrough and the wash from the column iscombined and measured with a scintillation counter. The degree ofphosphodiesterase inhibition is determined by calculating the amount ofradioactivity in drug-treated reactions and comparing against a controlsample (a reaction mixture lacking the tested compound but with drugsolvent).

Alternatively, the ability of desirable compounds to inhibit thephosphodiesterases of this invention is reflected by an increase in cGMPin neoplastic cells exposed to a compound being screened. The amount ofPDE activity can be determined by assaying for the amount of cyclic GMPin the extract of treated cells using radioimmunoassay (RIA). In thisprocedure, HT-29 or SW-480 cells are plated and grown to confluency. Asindicated above, SW-480 contains both PDE5 and the novel PDE of thisinvention, so when PDE activity is evaluated in this fashion, a combinedcGMP hydrolytic activity is assayed simultaneously. The test compound isthen incubated with the cell culture at a concentration of compoundbetween about 200 μM to about 200 pM. About 24 to 48 hours thereafter,the culture media is removed from the cells, and the cells aresolubilized. The reaction is stopped by using 0.2N HCl/50% MeOH. Asample is removed for protein assay. Cyclic GMP is purified from theacid/alcohol extracts of cells using anion-exchange chromatography, suchas a Dowex column. The cGMP is dried, acetylated according to publishedprocedures, such as using acetic anhydride in triethylamine, (Steiner,A. L., Parker, C. W., Kipnis, D. M., J. Biol. Chem., 247(4):1106-13,1971, which is incorporated herein by reference). The acetylated cGMP isquantitated using radioimmunoassay procedures (Harper, J., Brooker, G.,Advances in Nucleotide Research, 10:1-33, 1979, which is incorporatedherein by reference). Iodinated ligands (tyrosine methyl ester) ofderivatized cyclic GMP are incubated with standards or unknowns in thepresence of antisera and appropriate buffers. Antiserum may be producedusing cyclic nucleotide-haptene directed techniques. The antiserum isfrom sheep injected with succinyl-cGMP-albumin conjugates and diluted1/20,000. Dose-interpolation and error analysis from standard curves areapplied as described previously (Seibert, A. F., Thompson, W. J.,Taylor, A., Wilbourn, W. H., Barnard, J. and Haynes, J., J. AppliedPhysiol., 72:389-395, 1992, which is incorporated herein by reference).

In addition, the culture media may be acidified, frozen (−70° C.) andalso analyzed for cGMP and cAMP.

In addition to observing increases in the content of cGMP in neoplasticcells caused by desirable compounds, decreases in content of cAMP havealso been observed. It has been observed that a particularly desirablecompound (i.e., one that selectively induces apoptosis in neoplasticcells, but not substantially in normal cells) follows a time courseconsistent with cGMP-specific PDE inhibition as one initial actionresulting in an increased cGMP content within minutes. Secondarily,treatment of neoplastic cells with a desirable anti-neoplastic compoundleads to decreased cAMP content within 24 hours. The intracellulartargets of drug actions are being studied further, but current datasupport the concept that the initial rise in cGMP content and thesubsequent fall in cAMP content precede apoptosis in neoplastic cellsexposed to desirable compounds.

The change in the ratio of the two cyclic nucleotides may be a moreaccurate tool for evaluating desirable cGMP-specific phosphodiesteraseinhibition activity of test compounds, rather than measuring only theabsolute value of cGMP, only cGMP-specific phosphodiesterase inhibition,or only the level of cGMP hydrolysis. In neoplastic cells not treatedwith anti-neoplastic compounds, the ratio of cGMP content/cAMP contentis in the 0.03-0.05 range (i.e., 300-500 fmol/mg protein cGMP contentover 6000-8000 fmol/mg protein cAMP content). After exposure todesirable anti-neoplastic compounds, that ratio increases several fold(preferably at least about a three-fold increase) as the result of aninitial increase in cyclic GMP and the later decrease in cyclic AMP.

Specifically, it has been observed that particularly desirable compoundsachieve an initial increase in cGMP content in treated neoplastic cellsto a level of cGMP greater than about 500 fmol/mg protein. In addition,particularly desirable compounds cause the later decrease in cAMPcontent in treated neoplastic cells to a level of cAMP less than about4000 fmol/mg protein.

To determine the content of cyclic AMP, radioimmunoassay techniquessimilar to those described above for cGMP are used. Basically, cyclicnucleotides are purified from acid/alcohol extracts of cells usinganion-exchange chromatography, dried, acetylated according to publishedprocedures and quantitated using radioimmunoassay procedures. lodinatedligands of derivatized cyclic AMP and cyclic GMP are incubated withstandards or unknowns in the presence of specific antisera andappropriate buffers.

Verification of the cyclic nucleotide content may be obtained bydetermining the turnover or accumulation of cyclic nucleotides in intactcells. To measure intact cell cAMP, ³H-adenine pre-labeling is usedaccording to published procedures (Whalin, M. E., Garrett Jr., R. L.,Thompson, W. J., and Strada, S. J. “Correlation of cell-free braincyclic nucleotide phosphodiesterase activities to cyclic AMP decay inintact brain slices”, Sec. Mess. and Phos. Protein Research, 12:311-325,1989, which is incorporated herein by reference). The procedure measuresflux of labeled ATP to cyclic AMP and can be used to estimate intactcell adenylate cyclase or cyclic nucleotide phosphodiesterase activitiesdepending upon the specific protocol. Cyclic GMP accumulation was toolow to be studied with intact cell pre-labeling according to publishedprocedures (Reynolds, P. E., S. J. Strada and W. J. Thompson, “CyclicGMP Accumulation In Pulmonary Microvascular Endothelial Cells MeasuredBy Intact Cell Prelabeling,” Life Sci., 60:909-918, 1997, which isincorporated herein by reference).

The PDE inhibitory activity effect of a compound can also be determinedfrom a tissue sample. Tissue biopsies from humans or tissues fromanesthetized animals are collected from subjects exposed to the testcompound. Briefly, a sample of tissue is homogenized in 500 μl of 6%TCA. A known amount of the homogenate is removed for protein analysis.The remaining homogenate is allowed to sit on ice for 20 minutes toallow for the protein to precipitate. Next, the homogenate iscentrifuged for 30 minutes at 15,000 g at 4° C. The supernatant isrecovered, and the pellet recovered. The supernatant is washed fourtimes with five volumes of water saturated diethyl ether. The upperether layer is discarded between each wash. The aqueous ether extract isdried in a speed vac. Once dried, the sample can be frozen for futureuse, or used immediately. The dried extract is dissolved in 500 μl ofassay buffer. The amount of cGMP-specific inhibition is determined byassaying for the amount of cyclic nucleotides using RIA procedures asdescribed above.

The amount of inhibition is determined by comparing the activity of thenovel PDE (or PDE2) in the presence and absence of the compound.Inhibition of the novel PDE activity (or PDE2) is indicative that thecompound is useful for treating neoplasia. Significant inhibitoryactivity greater than that of the benchmark, exisulind, preferablygreater than 50% at a concentration of 10 μM or below, is indicativethat a compound should be further evaluated for antineoplasticproperties. Preferably, the IC₅₀ value for the novel PDE inhibitionshould be less than 50 μM for the compound to be further considered forpotential use.

D. Determining Whether a Compound Reduces Tumor Cell Growth

In an alternate embodiment, the method of the present invention involvesfurther determining whether the compound reduces the growth of tumorcells. Various cell lines can be used in the sample depending on thetissue to be tested. For example, these cell lines include:SW-480—colonic adenocarcinoma; HT-29—colonic adenocarcinoma, A-427—lungadenocarcinoma carcinoma; MCF-7—breast adenocarcinoma; andUACC-375—melanoma line; and DU145—prostrate carcinoma. Cytotoxicity dataobtained using these cell lines are indicative of an inhibitory effecton neoplastic lesions. These cell lines are well characterized, and areused by the United States National Cancer Institute in their screeningprogram for new anti-cancer drugs.

A compound's ability to inhibit tumor cell growth can be measured usingthe HT-29 human colon carcinoma cell line obtained from ATCC. HT-29cells have previously been characterized as a relevant colon tumor cellculture model (Fogh, J., and Trempe, G. In: Human Tumor Cells in Vitro,J. Fogh (eds.), Plenum Press, New York, pp. 115-159, 1975). HT-29 cellsare maintained in RPMI media supplemented with 5% fetal bovine calfserum (Gemini Bioproducts, Inc., Carlsbad, Calif.) and 2 mm glutamine,and 1% antibiotic-antimycotic in a humidified atmosphere of 95% air and5% CO₂ at 37° C. Briefly, HT-29 cells are plated at a density of 500cells/well in 96 well microtiter plates and incubated for 24 hours at37° C. prior to the addition of compound. Each determination of cellnumber involved six replicates. After six days in culture, the cells arefixed by the addition of cold trichloroacetic acid to a finalconcentration of 10% and protein levels are measured using thesulforhodamine B (SRB) colorimetric protein stain assay as previouslydescribed by Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon,J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M.R., “New Colorimetric Assay For Anticancer-Drug Screening,” J. Natl.Cancer Inst. 82: 1107-1112, 1990, which is incorporated herein byreference.

In addition to the SRB assay, a number of other methods are available tomeasure growth inhibition and could be substituted for the SRB assay.These methods include counting viable cells following trypan bluestaining, labeling cells capable of DNA synthesis with BrdU orradiolabeled thymidine, neutral red staining of viable cells, or MTTstaining of viable cells.

Significant tumor cell growth inhibition greater than about 50% at adose of 100 μM or below is further indicative that the compound isuseful for treating neoplastic lesions. Preferably, an IC₅₀ value isdetermined and used for comparative purposes. This value is theconcentration of drug needed to inhibit tumor cell growth by 50%relative to the control. Preferably, the IC₅₀ value should be less than100 μM for the compound to be considered further for potential use fortreating neoplastic lesions.

E. Determining Whether a Compound Induces Apoptosis

In a second alternate embodiment, the screening method of the presentinvention further involves determining whether the compound inducesapoptosis in cultures of tumor cells.

Two distinct forms of cell death may be described by morphological andbiochemical criteria: necrosis and apoptosis. Necrosis is accompanied byincreased permeability of the plasma membrane; the cells swell and theplasma membrane ruptures within minutes. Apoptosis is characterized bymembrane blebbing, condensation of cytoplasm and the activation ofendogenous endonucleases.

Apoptosis occurs naturally during normal tissue turnover and duringembryonic development of organs and limbs. Apoptosis also is induced bycytotoxic T-lymphocytes and natural killer cells, by ionizing radiationand by certain chemotherapeutic drugs. Inappropriate regulation ofapoptosis is thought to play an important role in many pathologicalconditions including cancer, AIDS, or Alzheimer's disease, etc.Compounds can be screened for induction of apoptosis using cultures oftumor cells maintained under conditions as described above. Treatment ofcells with test compounds involves either pre- or post-confluentcultures and treatment for two to seven days at various concentrations.Apoptotic cells are measured in both the attached and “floating”compartments of the cultures. Both compartments are collected byremoving the supernatant, trypsinizing the attached cells, and combiningboth preparations following a centrifugation wash step (10 minutes, 2000rpm). The protocol for treating tumor cell cultures with sulindac andrelated compounds to obtain a significant amount of apoptosis has beendescribed in the literature. (See, Piazza, G. A., et al., CancerResearch, 55:3110-16, 1995, which is incorporated herein by reference).The novel features include collecting both floating and attached cells,identification of the optimal treatment times and dose range forobserving apoptosis, and identification of optimal cell cultureconditions.

Following treatment with a compound, cultures can be assayed forapoptosis and necrosis by florescent microscopy following labeling withacridine orange and ethidium bromide. The method for measuring apoptoticcell number has previously been described by Duke & Cohen,“Morphological And Biochemical Assays Of Apoptosis,” Current ProtocolsIn Immunology, Coligan et al., eds., 3.17.1-3.17.16 (1992), which isincorporated herein by reference.

For example, floating and attached cells can be collected bytrypsinization and washed three times in PBS. Aliquots of cells can becentrifuged. The pellet can then be re-suspended in media and a dyemixture containing acridine orange and ethidium bromide prepared in PBSand mixed gently. The mixture can then be placed on a microscope slideand examined for morphological features of apoptosis.

Apoptosis can also be quantified by measuring an increase in DNAfragmentation in cells that have been treated with test compounds.Commercial photometric EIA for the quantitative, in vitro determinationof cytoplasmic histone-associated-DNA-fragments (mono- andoligonucleosomes) are available (Cell Death Detection ELISA^(okys), Cat.No. 1,774,425, Boehringer Mannheim). The Boehringer Mannheim assay isbased on a sandwich-enzyme-immunoassay principle using mouse monoclonalantibodies directed against DNA and histones, respectively. This allowsthe specific determination of mono- and oligonucleosomes in thecytoplasmatic fraction of cell lysates.

According to the vendor, apoptosis is measured in the following fashion.The sample (cell-lysate) is placed into a streptavidin-coated microtiterplate (“MTP”). Subsequently, a mixture of anti-histone-biotin andanti-DNA peroxidase conjugate are added and incubated for two hours.During the incubation period, the anti-histone antibody binds to thehistone-component of the nucleosomes and simultaneously fixes theimmunocomplex to the streptavidin-coated MTP via its biotinylation.Additionally, the anti-DNA peroxidase antibody reacts with the DNAcomponent of the nucleosomes. After removal of unbound antibodies by awashing step, the amount of nucleosomes is quantified by the peroxidaseretained in the immunocomplex. Peroxidase is determined photometricallywith ABTS7 (2,2′-Azido-[3-ethylbenzthiazolin-sulfonate]) as substrate.

For example, SW-480 colon adenocarcinoma cells are plated in a 96-wellMTP at a density of 10,000 cells per well. Cells are then treated withtest compound, and allowed to incubate for 48 hours at 37° C. After theincubation, the MTP is centrifuged, and the supernatant is removed. Thecell pellet in each well is then resuspended in lysis buffer for 30minutes. The lysates are then centrifuged and aliquots of thesupernatant (i.e., the cytoplasmic fraction) are transferred into astreptavidin-coated MTP. Care is taken not to shake the lysed pellets(i.e. cell nuclei containing high molecular weight, unfragmented DNA) inthe MTP. Samples are then analyzed.

Fold stimulation (FS=OD_(max)/OD_(veh)), an indicator of apoptoticresponse, is determined for each compound tested at a givenconcentration. EC₅₀ values may also be determined by evaluating a seriesof concentrations of the test compound.

Statistically significant increases in apoptosis (i.e., greater than 2fold stimulation at a concentration of 100 μM) are further indicativethat the compound is useful for treating neoplastic lesions. Preferably,the EC₅₀ value for apoptotic activity should be less than 100 μM for thecompound to be further considered for potential use for treatingneoplastic lesions. EC₅₀ is herein defined as the concentration thatcauses 50% induction of apoptosis relative to vehicle treatment.

F. Mammary Gland Organ Culture Model Tests

Test compounds identified by the above methods can be tested forantineoplastic activity by their ability to inhibit the incidence ofpre-neoplastic lesions in a mammary gland organ culture system. Thismouse mammary gland organ culture technique has been successfully usedby other investigators to study the effects of known antineoplasticagents such as certain NSAIDs, retinoids, tamoxifen, selenium, andcertain natural products, and is useful for validation of the screeningmethod of the present invention.

For example, female BALB/c mice can be treated with a combination ofestradiol and progesterone daily, in order to prime the glands to beresponsive to hormones in vitro. The animals are sacrificed, andthoracic mammary glands are excised aseptically and incubated for tendays in growth media supplemented with insulin, prolactin,hydrocortisone, and aldosterone. DMBA (7,12-dimethylbenz(a)anthracene)is added to medium to induce the formation of premalignant lesions.Fully developed glands are then deprived of prolactin, hydrocortisone,and aldosterone, resulting in the regression of the glands but not thepre-malignant lesions.

The test compound is dissolved in DMSO and added to the culture mediafor the duration of the culture period. At the end of the cultureperiod, the glands are fixed in 10% formalin, stained with alum carmine,and mounted on glass slides. The incidence of forming mammary lesions isthe ratio of the glands with mammary lesions to glands without lesions.The incidence of mammary lesions in test compound treated glands iscompared with that of the untreated glands.

The extent of the area occupied by the mammary lesions can bequantitated by projecting an image of the gland onto a digitation pad.The area covered by the gland is traced on the pad and considered as100% of the area. The space covered by each of the non-regressedstructures is also outlined on the digitization pad and quantitated bythe computer.

G. cGMP-Specific PDE Inhibitors

The invention includes the use of cGMP-specific PDE inhibitors such asexisulind and(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride (also called CP461) in combination with a human epidermalgrowth factor receptor tyrosine kinase inhibitor to treat patients withcancer. The preparation, properties, and methods of use of cGMP-specificPDE inhibitors are more fully described below and in the referencescited herein and incorporated by reference herein. Preferably, thecGMP-specific PDE inhibitor is(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride.

H. Human Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

The present invention discloses the use of an inhibitor of humanepidermal growth factor receptor tyrosine kinase in combination with acGMP-specific PDE inhibitor to treat patients with neoplasia.Preferably, the inhibitor of human epidermal growth factor receptortyrosine kinase is Iressa. The preparation, properties, and methods ofuse of Iressa are more fully described below or in the references citedherein and incorporated by reference herein.

General Schemes for Producing c-GMP-Specific PDE Inhibitors

There are several general schemes for producing compounds useful in thisinvention (U.S. Pat. Nos. 5,948,779, 6,066,634, 6,166,053). One generalscheme (which has several sub-variations) involves the case where bothR₃ and R₄ are both hydrogen. This first scheme is described immediatelybelow in Scheme I. The other general scheme (which also has severalsub-variations) involves the case where at least one of R₃ and R₄ is amoiety other than hydrogen but within the scope of Formula I above. Thissecond scheme is described below as “Scheme II.”

The general scheme for preparing compounds where both R₃ and R₄ are bothhydrogen is illustrated in Scheme I, which is described in part in U.S.Pat. No. 3,312,730, which is incorporated herein by reference. In SchemeI, R₁ is as defined in Formula I above. However, in Scheme I, thatsubstituent can also be a reactive moiety (e.g. a nitro group) thatlater can be reacted to make a large number of other substituted indenesfrom the nitro-substituted indenes.

In Scheme I, several sub-variations can be used. In one sub-variation, asubstituted benzaldehyde (a) may be condensed with a substituted aceticester in a Knoevenagel reaction (see reaction 2) or with an α-halogenopropionic ester in a Reformatsky Reaction (see reactions 1 and 3). Theresulting unsaturated ester (c) is hydrogenated and hydrolyzed to give asubstituted benzyl propionic acid (e) (see reactions 4 and 5).Alternatively, a substituted malonic ester in a typical malonic estersynthesis (see reactions 6 and 7) and hydrolysis decarboxylation of theresulting substituted ester (g) yields the benzyl propionic acid (e)directly. This latter method is especially preferable for nitro andalkylthio substituents on the benzene ring.

The next step is the ring closure of the β-aryl proponic acid (e) toform an indanone (h) which may be carried out by a Friedel-CraftsReaction using a Lewis acid catalyst (Cf. Organic Reactions, Vol. 2, p.130) or by heating with polyphosphoric acid (see reactions 8 and 9,respectively). The indanone (h) may be condensed with an α-halo ester inthe Reformatsky Reaction to introduce the aliphatic acid side chain byreplacing the carboxyl group (see reaction 10). Alternately, thisintroduction can be carried out by the use of a Wittig Reaction in whichthe reagent is a α-triphenylphosphinyl ester, a reagent which replacesthe carbonyl with a double bond to the carbon (see reaction 12). Thisproduct (1) is then immediately rearranged into the indene (j)(seereaction 13). If the Reformatsky Reaction route is used, theintermediate 3-hydroxy-3-aliphatic acid derivative i must be dehydratedto the indene (j) (see reaction 11).

The indenylacetic acid (k) in THF then is allowed to react with oxalylor thionyl chloride or similar reagent to produce the acid chloride (m)(see reaction 15), whereupon the solvent is evaporated. There are twomethods to carry out reaction 16, which is the addition of thebenzylamine side chain (n).

Method (I)

In the first method, the benzylamine (n) is added slowly at roomtemperature to a solution of 5-fluoro-2-methyl-3-indenylacetyl chloridein CH₂Cl₂. The reaction mixture is refluxed overnight, and extractedwith aqueous HCl (10%), water, and aqueous NaHCO₃ (5%). The organicphase is dried (Na₂SO₄) and is evaporated to give the amide compound (o)

Method (II)

In the second method, the indenylacetic acid (k) in DMA is allowed toreact with a carbodiimide (e.g.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) andbenzylamine at room temperature for two days. The reaction mixture isadded dropwise to stirred ice water. A yellow precipitate is filteredoff, is washed with water, and is dried in vacuo. Recrystallizationgives the amide compound (o).

Compounds of the type a′ (Scheme III), o (Scheme I), t (Scheme II), y(Scheme IIB) may all be used in the condensation reaction shown inScheme III.

Substituents

X=halogen, usually Cl or Br.

E=methyl, ethyl or benzyl, or lower acyl.

R₁, R₂, R₆, R₅, and R₇=as defined in Formula I.

Y, n and m=as defined in Formula I.

Reagents and general conditions for Scheme I (numbers refer to thenumbered reactions):

-   -   (1) Zn dust in anhydrous inert solvent such as benzene and        ether.    -   (2) KHSO₄ or p-toluene sulfonic acid.    -   (3) NaOC₂H₅ in anhydrous ethanol at room temperature.    -   (4) H₂ palladium on charcoal, 40 p.s.i. room temperature.    -   (5) NaOH in aqueous alcohol at 20-100°.    -   (6) NaOC₂H₅ or any other strong base such as NaH or        K-t-butoxide.    -   (7) Acid.    -   (8) Friedel-Crafts Reaction using a Lewis Acid catalyst Cf.        Organic Reactions, Vol. II, p. 130.    -   (9) Heat with polyphosphoric acid.    -   (10) Reformatsky Reaction: Zn in inert solvent, heat.    -   (11) p-Toluene sulfonic acid and CaCl₂ or I₂ at 200°    -   (12) Wittig Reaction using (C₆H₅)₃ P═C—COOE 20-80° in ether or        benzene    -   (13) (a) NBS/CCl₄/benzoyl peroxide        -   (b) PtO₂/H₂ (1 atm.)/acetic acid    -   (14) (a) NaOH        -   (b) HCl    -   (15) Oxalyl or thionyl chloride in CH₂Cl₂ or THF    -   (16) Method I: 2 equivalents of NH₂—C(R₅R₆)-Ph-(R₇)_(m)        -   Method II: carbodiimide in THF    -   (17) 1N NaOCH₃ in MeOH under reflux conditions

Indanones within the scope of compound (h) in Scheme I are known in theliterature and are thus readily available as intermediates for theremainder of the synthesis so that reactions 1-7 can be convenientlyavoided. Among such known indanones are:

-   -   5-methoxyindanone    -   6-methoxyindanone    -   5-methylindanone    -   5-methyl-6-methoxyindanone    -   5-methyl-7-chloroindanone    -   4-methoxy-7-chloroindanone    -   4-isopropyl-2,7-dimethylindanone    -   5,6,7-trichloroindanone    -   2-n-butylindanone    -   5-methylthioindanone

Scheme II has two mutually exclusive sub-schemes: Scheme IIA and SchemeII B. Scheme II A is used when R₃ is hydroxy and R₄ is hydrogen or whenthe two substituents form an oxo group. When R₃ is lower alkyl amino,Scheme II B is employed.

Similar to Scheme I, in Scheme IIA the indenylacetic acid (k) in THF isallowed to react with oxalylchloride under reflux conditions to producethe acid chloride (p) (see reaction 18), whereupon the solvent isevaporated. In reaction 19, a 0° C. mixture of a benzyl hydroxylaminehydrochloride (q) and Et₃N is treated with a cold solution of the acidchloride in CH₂Cl₂ over a period of 45-60 minutes. The mixture is warmedto room temperature and stirred for one hour, and is treated with water.The resulting organic layer is washed with 1 N HCl and brine, is driedover magnesium sulfate and is evaporated. The crude product, aN-hydroxy-N-benzyl acetamide (r) is purified by crystallization or flashchromatography. This general procedure is taught by Hoffman et al., JOC1992, 57, 5700-5707.

The next step is the preparation of the N-mesyloxy amide (s) in reaction20, which is also taught by Hoffman et al., JOC 1992, 57, 5700-5707.Specifically, to a solution of the hydroxamic acid (r) in CH₂Cl₂ at 0°C. is added triethylamine. The mixture is stirred for 10-12 minutes, andmethanesulfonyl chloride is added dropwise. The mixture is stirred at 0°C. for two hours, is allowed to warm to room temperature, and is stirredfor another two hours. The organic layer is washed with water, 1 N HCl,and brine, and is dried over magnesium sulfate. After rotaryevaporation, the product(s) is usually purified by crystallization orflash chromatography.

The preparation of the N-benzyl-α-(hydroxy) amide (t) in reaction 21, isalso taught by Hoffman et al., JOC 1992, 57, 5700-5707 and Hoffman etal., JOC 1995, 60, 4121-4125. Specifically, to a solution of theN-(mesyloxy) amide (s) in CH₃CN/H₂O is added triethylamine in CH₃CN overa period of 6-12 hours. The mixture is stirred overnight. The solvent isremoved, and the residue is dissolved in ethyl acetate. The solution iswashed with water, 1 N HCl, and brine, and is dried over magnesiumsulfate. After rotary evaporation, the product (t) is usually purifiedby recrystallization.

Reaction 22 in Scheme IIA involves a condensation with certainaldehydes, which is described in Scheme III below, a scheme that iscommon to products made in accordance with Schemes I, IIA and IIB.

The final reaction 23 in Scheme IIA is the preparation of theN-benzyl-α-ketoamide (v), which involves the oxidation of a secondaryalcohol (u) to a ketone by e.g. a Pfitzner-Moffatt oxidation, whichselectively oxidizes the alcohol

without oxidizing the Y group. Compounds (u) and (v) may be derivatizedin order to obtain compounds with R₃ and R₄ groups as set forth inFormula I.

As explained above, Scheme IIB is employed when R₃ is lower alkyl amino.Similar to Scheme I, in Scheme IIB the indenylacetic acid (k) in THF isallowed to react with oxalylchloride under reflux conditions to producethe acid chloride (p) (see reaction 18), whereupon the solvent isevaporated. In reaction 24, a mixture of an alkyl hydroxylaminehydrochloride (i.e. HO—NHR where R is a lower alky, preferablyisopropyl) and Et₃N is treated at 0° C. with a cold solution of the acidchloride in CH₂Cl₂ over a period of 45-60 minutes. The mixture is warmedto room temperature and is stirred for one hour, and is diluted withwater. The resulting organic layer is washed with 1 N HCl and brine, isdried over magnesium sulfate and is evaporated. The crude product, aN-hydroxy-N-alkyl acetamide (w) is purified by crystallization or flashchromatography. This general procedure is also taught by Hoffman et al.,JOC 1992, 57, 5700-5707

The preparation of the N-mesyloxy amide (x) in reaction 25, which isalso taught by Hoffman et al., JOC 1992, 57, 5700-5707. Specifically, asolution of the hydroxamic acid (w) in CH₂Cl₂ at 0° C. is treated withtriethylamine, is stirred for 10-12 minutes, and is treated dropwisewith methanesulfonyl chloride. The mixture is stirred at 0° C. for twohours, is allowed to warm to room temperature, and is stirred foranother two hours. The resulting organic layer is washed with water, 1 NHCl, and brine, and is dried over magnesium sulfate. After rotaryevaporation, the product (x) is usually purified by crystallization orflash chromatography.

The preparation of the N-benzyl indenyl-α-loweralkylamino-acetamidecompound (y) in Scheme IIB as taught by Hoffinan et al., JOC 1995, 60,4121-25 and J. Am. Chem Soc. 1993, 115, 5031-34, involves the reactionof the N-mesyloxy amide (x), with a benzylamine in CH₂Cl₂ at 0° C. isadded over a period of 30 minutes. The resulting solution is stirred at0° C. for one hour and at room temperature overnight. The solvent isremoved, and the residue is treated with 1 N NaOH. The extract withCH₂Cl₂ is washed with water and is dried over magnesium sulfate. Afterrotary evaporation, the product (y) is purified by flash chromatographyor crystallization.

Scheme III involves the condensation of the heterocycloaldehydes (i.e.Y—CHO) with the indenyl amides to produce the final compounds of FormulaI. This condensation is employed, for example, in reaction 17 in SchemeI above and in reaction 22 in Scheme IIA. It is also used to convertcompound (y) in Scheme IIB to final compounds of Formula I.

In Scheme III, the amide (a′) from the above schemes, aN-heterocycloaldehyde (z), and sodium methoxide (1 M in methanol) arestirred at 60° C. under nitrogen for 24 hours. After cooling, thereaction mixture is poured into ice water. A solid is filtered off, iswashed with water, and is dried in vacuo. Recrystallization provides acompound of Formula I in Schemes I and IIB and the intermediate (u) inScheme IIA.

As has been pointed out above, it is preferable in the preparation ofmany types of the compounds of this invention, to use a nitrosubstituent on the benzene ring of the indanone nucleus and convert itlater to a desired substituent since by this route a great manysubstituents can be reached. This is done by reduction of the nitro tothe amino group followed by use of the Sandmeyer Reaction to introducechlorine, bromine, cyano or xanthate in place of the amino. From thecyano derivatives hydrolysis yields the carboxamide and carboxylic acid;other derivatives of the carboxy group such as the esters can then beprepared. The xanthates, by hydrolysis, yield the mercapto group thatmay be oxidized readily to the sulfonic acid or alkylated to analkylthio group which can then be oxidized to alkylsulfonyl groups.These reactions may be carried out either before or after theintroduction of the 1-substituent.

Based on the above-identified schema, the following are examples ofcompounds of the invention and their production and identification:

EXAMPLE 1(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-Indenylacetamide

(A) p-Fluoro-α-methylcinnamic acid

p-Fluorobenzaldehyde (200 g, 1.61 mol), propionic anhydride (3.5 g, 2.42mol) and sodium propionate (155 g, 1.61 mol) are mixed in a one literthree-necked flask which had been flushed with nitrogen. The flask isheated gradually in an oil-bath to 140° C. After 20 hours, the flask iscooled to 100° C. and poured into 8 l of water. The precipitate isdissolved by adding potassium hydroxide (302 g) in 2 l of water. Theaqueous solution is extracted with ether, and the ether extracts arewashed with potassium hydroxide solution. The combined aqueous layersare filtered, are acidified with concentrated HCl, and are filtered. Thecollected solid, p-fluoro-α-methylcinnamic acid, is washed with water,and is dried and used as obtained.

(B) p-Fluoro-α-methylhydrocinnamic acid

To p-fluoro-α-methylcinnamic acid (177.9 g, 0.987 mol) in 3.6 l ethanolis added 11.0 g of 5% Pd/C. The mixture is reduced at room temperatureunder a hydrogen pressure of 40 p.s.i. When hydrogen uptake ceases, thecatalyst is filtered off, and the solvent is evaporated in vacuo to givethe product, p-fluoro-α-methylhydrocinnamic acid, which was useddirectly in the next step.

(C) 6-Fluoro-2-methylindanone

To 932 g polyphosphoric acid at 70° C. (steam bath) is addedp-fluoro-α-methylhydrocinnamic acid (93.2 g, 0.5 mol) slowly withstirring. The temperature is gradually raised to 95° C., and the mixtureis kept at this temperature for 1 hour. The mixture is allowed to cooland is added to 2 l. of water. The aqueous suspension is extracted withether. The extract is washed twice with saturated sodium chloridesolution, 5% Na₂CO₃ solution, and water, and is dried, and isconcentrated on 200 g silica-gel; the slurry is added to a five poundsilica-gel column packed with 5% ether-petroleum ether. The column iseluted with 5-10% ether-petroleum ether, to give6-fluoro-2-methylindanone. Elution is followed by TLC.

(D) 5-fluoro-2-methylindenyl-3-acetic acid

A mixture of 6-fluoro-2-methylindanone (18.4 g, 0.112 mol), cyanoaceticacid (10.5 g, 0.123 mol), acetic acid (6.6 g), and ammonium acetate (1.7g) in dry toluene (15.5 ml) is refluxed with stirring for 21 hours, asthe liberated water is collected in a Dean Stark trap. The toluene isevaporated, and the residue is dissolved in 60 ml of hot ethanol and 14ml of 2.2 N aqueous potassium hydroxide solution. 22 g of 85% KOH in 150ml of water is added, and the mixture refluxed for 13 hours undernitrogen. The ethanol is removed under vacuum, and 500 ml water isadded. The aqueous solution is extracted well with ether, and is thenboiled with charcoal. The aqueous filtrate is acidified to pH 2 with 50%cold hydrochloric acid. The precipitate is dried and5-fluoro-2-methylindenyl-3-acetic acid (M.P. 164-166° C.) is obtained.

(E) 5-fluoro-2-methylindenyl-3-acetyl chloride

5-fluoro-2-methylindenyl-3-acetic acid (70 mmol) in THF (70 ml) isallowed to react with oxalylchloride (2 M in CH₂Cl₂; 35 ml; 70 mmol)under reflux conditions (24 hours). The solvent is evaporated to yieldthe title compound, which is used as such in the next step.

(F) 5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide

Benzylamine (5 mmol) is added slowly at room temperature to a solutionof 5-fluoro-2-methylindenyl-3-acetyl chloride (2.5 mmol.) in CH₂Cl₂ (10ml). The reaction mixture is refluxed overnight, and is extracted withaqueous HCl (10%), water, and aqueous NaHCO₃ (5%). The organic phase isdried (Na₂SO₄) and is evaporated to give the title compound, which isrecrystallized from CH₂Cl₂ to give the title compound as a white solid(m.p. 144° C.).

(G)(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (3.38 mmol),4-pyridinecarboxaldehyde (4 mmol), sodium methoxide (1M NaOCH₃ inmethanol (30 ml)) are heated at 60° C. under nitrogen with stirring for24 hours. After cooling, the reaction mixture is poured into ice water(200 ml). A solid is filtered off, washed with water, and dried invacuo. Recrystallization from CH₃CN gives the title compound (m.p. 202°C.) as a yellow solid (R₁=F, R₂=CH₃, R₃=H, R₄=H, R₅=H, R₆=H, R₇=H, n=1,m=1, Y=4-pyridinyl).

(H)(E)-5-Fluoro-2-methyl-(4-pyridinlidene)-3-(N-benzyl)-indenylacetamide

The mother liquor obtained from the CH₃CN recrystallization of 1G isrich on the geometrical isomer of 1G. The E-isomer can be obtained pureby repeated recrystallizations from CH₃CN.

EXAMPLE 2(Z)-5-Fluoro-2-Methyl-(3-Pyridinylidene)-3-(N-Benzyl)-Indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 3-pyridinecarboxaldehyde. Recrystallization from CH₃CN gives thetitle compound (m.p. 175° C.)(R₁=F, R₂=CH₃, R₃=H, R₄=H, R₅=H, R₆=H,R₇=H, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 3(Z)-5-Fluoro-2-Methyl-(2-Pyridinylidene)-3-(N-Benzyl)-Indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 2-pyridinecarboxaldehyde. Recrystallization from ethylacetate givesthe title compound (m.p. 218° C.)(R₁=F, R₂=CH₃, R₃=H, R₄=H, R₅=H, R₆=H,R₇=H, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 4(Z)-5-Fluoro-2-Methyl-(4-Quinolinylidene)-3-(N-Benzyl)-Indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 4-quinolinecarboxaldehyde. Recrystallization from ethylacetategives the title compound (m.p. 239° C.)(R₁=F, R₂=CH₃, R₃=H, R₄=H, R₅=H,R₆=H, R₇=H, n=1, m=1, Y=4-quinolinyl).

EXAMPLE 5(Z)-5-Fluoro-2-Methyl-(4,6-Dimethyl-2-Pyridinylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 4,6-dimethyl-2-pyridinecarboxaldehyde accordingto the procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4,6-dimethyl-2-pyridinyl).

EXAMPLE 6(Z)-5-Fluoro-2-Methyl-(3-Quinolinylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 3-quinolinecarboxaldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=3-quinolinyl)

EXAMPLE 7(Z)-5-Fluoro-2-Methyl-(2-Quinolinylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 2-quinolinecarboxaldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=2-quinolinyl).

EXAMPLE 8(Z)-5-Fluoro-2-Methyl-(Pyrazinylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyrazinealdehyde according to the procedure ofExample 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=pyrazinyl).

EXAMPLE 9(Z)-5-Fluoro-2-Methyl-(3-Pyridazinylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyridazine-3-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 10(Z)-5-Fluoro-2-Methyl-(4-Pyrimidinylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyrimidine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4-pyrimidinyl).

EXAMPLE 11(Z)-5-Fluoro-2-Methyl-(2-Methyl-4-Pyrimidinylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 2-methyl-pyrimidine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=2-methyl-4-pyrimidinyl).

EXAMPLE 12(Z)-5-Fluoro-2-Methyl-(4-Pyridazinylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyridazine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 13(Z)-5-Fluoro-2-Methyl-(1-Methyl-3-Indolylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 1-methylindole-3-carboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=1-methyl-3-indolyl).

EXAMPLE 14(Z)-5-Fluoro-2-Methyl-(1-Acetyl-3-Indolylidene)-3-(N-Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 1-acetyl-3-indolecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=1-acetyl-3-indolyl).

EXAMPLE 15(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-2-Fluorobenzyl)-Indenylacetamide

(A) 5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide

This compound is obtained from 5-fluoro-2-methylindenyl-3-acetylchloride (Example 1E) using the procedure of Example 1, Part F andreplacing benzylamine with 2-fluorobenzylamine.

(B)(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-2-fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide is allowed toreact with 4-pryidinecarboxaldehyde according to the procedure ofExample 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=F, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 16(Z)-5-Fluoro-2-Methyl-(3-Pyridinylidene)-3-(N-2-Fluorobenzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 3-pryidinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=F, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 17(Z)-5-Fluoro-2-Methyl-(2-Pyridinylidene)-3-(N-2-Fluorobenzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 2-pyridinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=F, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 18(Z)-5-Fluoro-2-Methyl-(4-Quinolinylidene)-3-(N-2-Fluorobenzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 4-quinolinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=F, n=1, m=1, Y=3-quinolinyl).

EXAMPLE 19(Z)-5-Fluoro-2-Methyl-(3-Pyrazinylidene)-3-(N-2-Fluorobenzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pyrazinealdehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=F, n=1, m=1, Y=3-pyrazinyl).

EXAMPLE 20(Z)-5-Fluoro-2-Methyl-(3-Pyridazinylidene)-3-(N-2-Fluorobenzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 3-pryidaziine-3-aldehyde according tothe procedure of Example 1, Part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=H, R₆=H, R₇=F, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 21(Z)-5-Fluoro-2-Methyl-(3-Pyrimidinylidene)-3-(N-2-Fluorobenzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pryimidine-4-aldehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=F, n=1, m=1, Y=3-pyrimidinyl).

EXAMPLE 22(Z)-5-Fluoro-2-Methyl-(4-Pyridazinylidene)-3-(N-2-Fluorobenzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pryidazine-4-aldehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁=F, R₂=CH₃, R₃=H, R₄=H,R₅=H, R₆=H, R₇=F, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 23(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-Indenylacetamide

(A) 5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide

5-Fluoro-2-methylindenyl-3-acetic acid (from Example 1D) (2.6 mmol) inDMA (2 ml) is allowed to react withn-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (4 mmol)and S-2-amino-2-phenylethanol (3.5 mmol) at room temperature for twodays. The reaction mixture is added dropwise to stirred ice water (50ml). A white precipitate is filtered off, washed with water (5 ml), anddried in vacuo. Recrystallization from ethylacetate gives the desiredcompound.

(B)(Z)-5-fluoro-2-methyl-(4-pyridinylidene)-3-(N-(S-α-hydroxymethyl)benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide frompart A is allowed to react with 4-pryidinecarboxaldehyde according tothe procedure of Example 1, Part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁=F, R₂=CH₃,R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 24(Z)-5-Fluoro-2-Methyl-(3-Pyridinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 3-pryidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 25(Z)-5-Fluoro-2-Methyl-(2-Pyridinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 2-pryidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1 , Y=2-pyridinyl).

EXAMPLE 26(Z)-5-Fluoro-2-Methyl-(4-Quinolinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 4-quinolinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=4-quinolinyl).

EXAMPLE 27(Z)-5-Fluoro-2-Methyl-(Pyrazidinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryazidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=pyrazidinyl).

EXAMPLE 28(Z)-5-Fluoro-2-Methyl-(3-Pyridazinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryidazine-3-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 29(Z)-5-Fluoro-2-Methyl-(4-Pyrimidinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryimidine-4-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=4-pyrimidinyl).

EXAMPLE 30(Z)-5-Fluoro-2-Methyl-(4-Pyridazinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-Indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryidazine-4-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁=F,R₂=CH₃, R₃=H, R₄=H, R₅=CH₂OH, R₆=H, R₇=H, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 31rac-(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)Indenyl-α-Hydroxyacetamide

(A) 5-fluoro-2-methyl-3-(N-benzyl-N-hydroxy)-indenylacetamide

To a mixture of N-benzylhydroxylamine hydrochoride (12 mmol) and Et₃N(22 mmol) in CH₂Cl₂ (100 ml) at 0° C. is added a cold solution of5-fluoro-2-methylindenyl-3-acetyl chloride (Example 1, Step E) (10 mmol)in CH₂Cl₂ (75 ml) over a period of 45-60 minutes. The mixture is warmedto room temperature and is stirred for 1 hour. The mixture is dilutedwith water (100 ml), and the organic layer is washed with HCl (2×25 ml)and brine (2×100 ml), dried (MgSO₄) and evaporated. The crude product ispurified with flash chromatography to give the title compound.

(B) 5-Fluoro-2-methyl-3-(N-benzyl-N-mesyloxy)-indenylacetamide

To a solution of5-fluoro-2-methyl-3-(N-benzyl-N-hydroxy)-indenylacetamide (5 mmol) inCH₂Cl₂ (25 ml) at 0° C. is added triethylamine (5 mmol). The mixture isstirred for 10 minutes, and methanesulfonyl chloride (5.5 mmol) is addeddropwise. The solution is stirred at 0° C. for 2 hours, allowed to warmto room temperature, and stirred for another 2 hours. The organic layeris washed with water (2×20 ml), in HCl (15 ml), and brine (20 ml) anddried over MgSO₄. After rotary evaporation, the product is purified withflash chromatography to give the title compound.

(C) rac-5-Fluoro-2-methyl-3-(N-benzyl)-α-hydroxyindenylacetamide

To a solution of5-fluoro-2-methyl-3-(N-benzyl-N-mesyloxy)-indenylacetamide (2 mmol) inCH₃CN/H₂O (12 ml. each) is added triethylamine (2.1 mmol) in CH₃CN (24ml) over a period of 6 hours. The mixture is stirred overnight. Thesolvent is removed, and the residue diluted with ethyl acetate (60 ml),washed with water (4×20 ml), in HCl (15 ml), and brine (20 ml) and driedover MgSO₄. After rotary evaporation, the product is purified byrecrystallization to give the title compound.

(D)rac-(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-hydroxyacetamideis obtained fromrac-5-fluoro-2-methyl-3-(N-benzyl)-α-hydroxyindenylacetamide using theprocedure of Example 1, Part G (R₁=F, R₂=CH₃, R₃=OH, R₄=H, R₅=H, R₆=H,R₇=H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 322-[(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-Indenyl]-Oxyacetamide

For Pfitzner-Moffatt oxidation, a solution ofrac-(Z)-5-fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-hydroxyacetamide(1 mmol) in DMSO (5 ml) is treated with dicyclohexylcarbodiimide (3mmol). The mixture is stirred overnight, and the solvent is evaporated.The crude product is purified by flash chromatography to give the titlecompound (R₁=F, R₂=CH₃, R₃ and R₄ together form C═O, R₅=H, R₆=H, R₇=H,n=1, m=1, and Y=4-pyridinyl).

EXAMPLE 33

rac-(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-Indenyl-α-(2-Propylamino)-Acetamide

(A) 5-Fluoro-2-methyl-3-(N-2-propyl-N-hydroxy)-indenylacetamide isobtained from 5-fluoro-2-methylindenyl-3-acetyl chloride (Example 1,Step E) using the procedure of Example 31, Part A and replacingN-benzylhydroxylamine hydrochloride with N-2-propyl hydroxylaminehydrochloride.

(B) 5-Fluoro-2-methyl-3-(N-2-propyl-N-mesyloxy)-indenylacetamide isobtained according to the procedure of Example 31, Part B.

(C) rac-5-Fluoro-2-methyl-3-(N)-benzyl)-α-(2-propylamino)-acetamide. To5-fluoro-2-methyl-3-(N-2-propyl-N-mesyloxy)-indenylacetamide (2 mmol) inCH₂Cl₂ (25 ml) at 0° C. is added benzylamine (4.4 mmol) in CH₂Cl₂ (15ml) over a period of 30 minutes. The resulting solution is stirred at 0°C. for 1 hour, and at room temperature overnight. The solvent isremoved, and the residue is treated with 1 N NaOH, and is extracted withCH₂Cl₂ (100 ml). The extract is washed with water (2×10 ml), and isdried over MgSO₄. After rotary evaporation, the product is purified byflash chromatography.

(D)rac-(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-(2-propylamino)-acetamideis obtained fromrac-5-fluoro-2-methyl-3-(N-benzyl)-α-(2-propylamino)-acetamide using theprocedure of Example 1, Part G (R₁=F, R₂=CH₃, R₃=isopropylamino, R₄=H,R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 34(Z)-6-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-Indenylacetamide

(A) Ethyl-2-Hydroxy-2-(p-Methoxyphenyl)-1-Methylpropionate

In a 500 ml. 3-necked flask is placed 36.2 g. (0.55 mole) of zinc dust,a 250 ml. addition funnel is charged with a solution of 80 ml. anhydrousbenzene, 20 ml. of anhydrous ether, 80 g. (0.58 mole) of p-anisaldehydeand 98 g. (0.55 mole) of ethyl-2-bromopropionate. About 10 ml. of thesolution is added to the zinc dust with vigorous stirring, and themixture is warmed gently until an exothermic reaction commences. Theremainder is added dropwise at such a rate that the reaction mixturecontinues to reflux smoothly (ca. 30-35 min.). After addition iscompleted the mixture is placed in a water bath and refluxed for 30minutes. After cooling to 0°, 250 ml. of 10% sulfuric acid is added withvigorous stirring. The benzene layer is extracted twice with 50 ml.portions of 5% sulfuric acid and washed twice with 50 ml. portions ofwater. The combined aqueous acidic layers are extracted with 2×50 ml.ether. The combined etheral and benzene extracts are dried over sodiumsulfate. Evaporation of solvent and fractionation of the residue througha 6″ Vigreux column affords 89 g. (60%) of the product,ethyl-2-hydroxy-2-(p-methoxyphenyl)-1-methylpropionate, B.P. 165-160°(1.5 mm.).

(B) 6-Methoxy-2-methylindanone

By the method described in Vander Zanden, Rec. Trav. Chim., 68, 413(1949), the compound from part A is converted to6-methoxy-2-methylindanone.

Alternatively, the same compound can be obtained by addingα-methyl-β-(p-methoxylphenyl)propionic acid (15 g.) to 170 g. ofpolyphosphoric acid at 50° and heating the mixture at 83-90° for twohours. The syrup is poured into iced water. The mixture is stirred forone-half hour, and is extracted with ether (3×). The etheral solution iswashed with water (2×) and 5% NaHCO₃ (5×) until all acidic material hasbeen removed, and is dried over sodium sulfate. Evaporation of thesolution gives 9.1 g. of the indanone as a pale yellow oil.

(C)(Z)-6-Methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

In accordance with the procedures described in Example 1, parts D-G,this compound is obtained substituting 6-methoxy-2-methylindanone for6-fluoro-2-methylindanone in part D of Example 1.

EXAMPLE 35(Z)-5-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-Indenylacetamide

(A) Ethyl 5-Methoxy-2-Methyl-3-Indenyl Acetate

A solution of 13.4 g of 6-methoxy-2-methylindanone and 21 g. of ethylbromoacetate in 45 ml. benzene is added over a period of five minutes to21 g. of zinc amalgam (prepared according to Org. Syn. Coll. Vol. 3) in110 ml. benzene and 40 ml. dry ether. A few crystals of iodine are addedto start the reaction, and the reaction mixture is maintained at refluxtemperature (ca. 65°) with external heating. At three-hour intervals,two batches of 10 g. zinc amalgam and 10 g. bromoester are added and themixture is then refluxed for 8 hours. After addition of 30 ml. ofethanol and 150 ml. of acetic acid, the mixture is poured into 700 ml.of 50% aqueous acetic acid. The organic layer is separated, and theaqueous layer is extracted twice with ether. The combined organic layersare washed thoroughly with water, ammonium hydroxide and water. Dryingover sodium sulfate, evaporation of solvent in vacuo followed by pumpingat 80° (bath temperature)(1-2 mm.) gives crudeethyl-(1-hydroxy-2-methyl-6-methoxy-indenyl) acetate (ca. 18 g.).

A mixture of the above crude hydroxyester, 20 g. of p-toluenesulfonicacid monohydrate and 20 g. of anhydrous calcium chloride in 250 ml.toluene is refluxed overnight. The solution is filtered, and the solidresidue is washed with toluene. The combined toluene solution is washedwith water, sodium bicarbonate, water and then dried over sodiumsulfate. After evaporation, the crude ethyl 5-methoxy-2-methyl-3-indenylacetate is chromatographed on acid-washed alumina and the product iseluted with petroleum ether-ether (v./v. 50-100%) as a yellow oil (11.8g., 70%).

(B)(Z)-5-Methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenylacetamide

In accordance with the procedures described in Example 1, parts E-G,this compound is obtained substitutingethyl-5-methoxy-2-methyl-3-indenyl acetate for5-fluoro-2-methindenyl-3-acetic acid in Example 1, part E.

EXAMPLE 36(Z)-α-5-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-Indenylpropionamide

(A) α-(5-Methoxy-2-methyl-3-indenyl)propionic acid

The procedure of Example 35, part (A) is followed using ethylα-bromopropionate in equivalent quantities in place of ethylbromoacetate used therein. There is obtained ethylα-(1-hydroxy-6-methoxy-2-methyl-1-indanyl)propionate, which isdehydrated to ethyl α-(5-methoxy-2-methyl-3-indenyl)propionate in thesame manner.

The above ester is saponified to giveα-(5-methoxy-2-methyl-3-indenyl)propionic acid.

(B) (Z)-α-5-Methoxy-2-methyl-(4-pyridinyl)-3-(N-benzyl)-α-methylindenylpropionamide

In accordance with the procedures described in Example 1, parts E-G,this compound is obtained substitutingα-5-methoxy-2-methyl-3-indenyl)propionic acid for5-fluoro-2-methylindenyl-3-acetic acid in Example 1, part E.

EXAMPLE 37 (Z)α-Fluoro-5-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)Indenylacetamide

(A) Methyl-5-Methoxy-2-Methyl-3-Indenyl-α-Fluoro Acetate

A mixture of potassium fluoride (0.1 mole) andmethyl-5-methoxy-2-methyl-3-indenyl-α-tosyloxy acetate (0.05 mole) in200 ml. dimethylformamide is heated under nitrogen at the refluxtemperature for 2-4 hours. The reaction mixture is cooled, poured intoiced water and then extracted with ether. The ethereal solution iswashed with water, sodium bicarbonate and dried over sodium sulfate.Evaporation of the solvent and chromatography of the residue on anacid-washed alumina column (300 g.) using ether-petroleum ether (v./v.20-50%) as eluent give the product,methyl-5-methoxy-2-methyl-3-indenyl-α-fluoroacetate.

(B) (Z)α-Fluoro-5-methoxy-2-methyl-(4-pyridinylidene)-3-(N-benzyl)indenylacetamide

In accordance with the procedures described in Example 1 above, partsE-G, this compound is obtained substitutingmethyl-5-methoxy-2-methyl-3-indenyl-α-fluoroacetate for5-fluoro-2-methylindenyl-3-acetic acid in Example 1 above, part E.

For the introduction of the ═CH—Y part in Scheme III, any of theappropriate heterocyclic aldehydes may be used either directly in thebase-catalyzed condensation or in a Wittig reaction in an alternativeroute. The aldehydes that may be used are listed in Table A below:

TABLE A pyrrol-2-aldehyde* pyrimidine-2-aldehyde6-methylpyridine-2-aldehyde* 1-methylbenzimidazole-2-aldehydeisoquinoline-4-aldehyde 4-pyridinecarboxaldehyde*3-pyridinecarboxaldehyde* 2-pyridinecarboxaldehyde*4,6-dimethyl-2-pyridinecarboxaldehyde* 4-methyl-pyridinecarboxaldehyde*4-quinolinecarboxaldehyde* 3-quinolinecarboxaldehyde*2-quinolinecarboxaldehyde* 2-chloro-3-quinolinecarboxaldehyde*pyrazinealdehyde (Prepared as described by Rutner et al., JOC 1963, 28,1898-99) pyridazine-3-aldehyde (Prepared as described by Heinisch etal., Monatshefte Fuer Chemie 108, 213-224, 1977) pyrimidine-4-aldehyde(Prepared as described by Bredereck et al., Chem. Ber. 1964, 97, 3407-17) 2-methyl-pyrimidine-4-aldehyde (Prepared as described by Brederecket al., Chem. Ber. 1964, 97, 3407- 17) pyridazine-4-aldehyde (Preparedas described by Heinisch et al., Monatshefte Fuer Chemie 104, 1372-1382(1973)) 1-methylindole-3-carboxaldehyde*1-acetyl-3-indolecarboxaldehyde* *Available from Aldrich

Available from Aldrich

The aldehydes above can be used in the reaction schemes above incombination with various appropriate amines to produce compounds withthe scope of this invention. Examples of appropriate amines are thoselisted in Table B below:

TABLE B benzylamine 2,4-dimethoxybenzylamine 2-methoxybenzylamine2-fluorobenzylamine 4-dimethylaminobenzylamine 4-sulfonaminobenzylamine1-phenylethylamine (R-enantiomer) 2-amino-2-phenylethanol (S-enantiomer)2-phenylglycinonitrile (S-enantiomer)

EXAMPLE 38(Z)-5-Fluoro-2-Methyl-(4-Pyridylidene)-3-(N-Benzyl)IndenylacetamideHydrochloride

(Z)-5-Fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamide(1396 g; MW 384.45; 3.63 mol) from Example 1 is dissolved at 45° C. inethanol (28 L). Aqueous HCl (12 M; 363 mL) is added stepwise. Thereaction mixture is heated under reflux for 1 hour, is allowed to coolto room temperature, then stored at −10° C. for 3 hours. The resultingsolid is filtered off, is washed with ether (2×1.5 L) and is air-driedovernight. Drying under vacuum at 70° C. for 3 days gives(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride with a melting point of 207-209° C. (R₁=F, R₂=CH₃, R₃=H,R₄=H, R₅=H, R₆=H, R₇=H, n=1, m=1, Y=4-pyridinyl.hydrochloride). Yield:1481 g (97%; 3.51 mol); MW: 420.91 g/mol. This compound is also known asCP461.

¹H-NMR (DMSO-d₆): 2.18 (s, 3, ═C—CH₃); 3.54 (s,2, ═CH₂CO); 4.28 (d, 2,NCH₂); 6.71 (m, 1, ar.); 7.17 (m, 8, ar.); 8.11 (d, 2, ar., AB system);8.85 (m, 1, NH); 8.95 (d, 2, ar., AB system); IR (KBr): 3432 NH; 1635C═O; 1598 C═C.

Experimental Results

A number of compounds were examined in the various protocols andscreened for potential use in treating neoplasia. The results of thesetests are reported below. The test compounds are hereinafter designatedby a letter code that corresponds to the following:

A - rac-threo-(E)-1-(N,N′-diethylaminoethanethio)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan; B -(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-acetic acid; C -(Z)-5-Fluoro-2-methyl-1-(p-chlorobenzylidene)-3-acetic acid; D -rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-N-acetylcysteine; E -(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetamide, N-benzyl; F -(Z)-5-Fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenylacetamide, N,N′-dicyclohexyl; G -ribo-(E)-1-Triazolo-[2′,3′:1″,3″]-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan; and H -rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-glutathione).

Experimental Example 1 COX Inhibition Assay

Reference compounds and test compounds were analyzed for their COXinhibitory activity in accordance with the protocol for the COX assay,supra. FIG. 4 shows the effect of various concentrations of eithersulindac sulfide or exisulind on purified cyclooxygenase (Type 1)activity. Cyclooxygenase activity was determined using purifiedcyclooxygenase from ram seminal vesicles as described previously(Mitchell et al, supra). The IC₅₀ value for sulindac sulfide wascalculated to be approximately 1.76 μM, while that for exisulind wasgreater than 10,000 μM. These data show that sulindac sulfide, but notexisulind, is a COX-I inhibitor. Similar data were obtained for theCOX-2 isoenzyme (Thompson, et al., Journal of the National CancerInstitute, 87: 1259-1260, 1995).

FIG. 5 shows the effect of test compounds B and E on COX inhibition. COXactivity was determined as for the compounds shown in FIG. 4. The datashow that neither test compound B and E significantly inhibits COX-I.

TABLE 2 Cyclooxygenase inhibitory activity for a series of compoundsReference compounds % Inhibition at 100 μM Indomethacin 95 MY5445 94Sulindac sulfide 97 Exisulind <25 Test compounds % Inhibition at 100 μMA <25 B <25 C 87 D <25 E <25

In accordance with the protocol, supra, compounds A through E wereevaluated for COX inhibitory activity as reported in Table 2 above.Compound C was found to inhibit COX greater than 25% at a 100 μM dose,and therefore, would not be selected for further screening.

Experimental Example 2 cGMP PDE Inhibition Assay

Reference compounds and test compounds were analyzed for their cGMP PDEinhibitory activity in accordance with the protocol for the assaydescribed supra. FIG. 6 shows the effect of various concentrations ofsulindac sulfide and exisulind on either PDE4 or cGMP PDE activitypurified from human colon HT-29 cultured tumor cells, as describedpreviously (W. J. Thompson et al., supra). The IC₅₀ value of sulindacsulfide for inhibition of PDE4 was 41 μM, and for inhibition of cGMP PDEwas 17 μM. The IC₅₀ value of exisulind for inhibition of PDE4 was 181μM, and for inhibition of cGMP PDE was 56 μM. These data show that bothsulindac sulfide and exisulind inhibit phosphodiesterase activity. Bothcompounds show selectivity for the cGMP PDE isoenzyme forms over PDE4isoforms.

FIG. 7 shows the effects of sulindac sulfide on either cGMP or cAMPproduction as determined in cultured HT-29 cells in accordance with theassay described, supra. HT-29 cells were treated with sulindac sulfidefor 30 minutes and cGMP or cAMP was measured by conventionalradioimmunoassay method. As indicated, sulindac sulfide increased thelevels of cGMP by greater than 50% with an EC₅₀ value of 7.3 μM (FIG.7A). Levels of cAMP were unaffected by treatment, although a known PDE4inhibitor, rolipram, increased cAMP (FIG. 7B). The data demonstrate thepharmacological significance of inhibiting cGMP PDE, relative to PDE4.

FIG. 8 shows the effect of the indicated dose of test compound B oneither cGMP PDE or PDE4 isozymes of phosphodiesterase. The calculatedIC₅₀ value was 18 μM for cGMP PDE and was 58 μM for PDE4.

FIG. 9 shows the effect of the indicated dose of test compound E oneither PDE4 or cGMP PDE. The calculated IC₅₀ value was 0.08 μM for cGMPPDE and greater than 25 μM for PDE4.

TABLE 3 cGMP PDE inhibitory activity among a series of compoundsReference compounds % Inhibition at 10 μM Indomethacin 34 MY5445 86Sulindac sulfide 97 Exisulind 39 Test compounds % Inhibition at 10 μM A<25 B <25 C <25 D 36 E 75

The above compounds in Table 3 were evaluated for PDE inhibitoryactivity, as described in the protocol supra. Of the compounds that didnot inhibit COX, only compound E was found to cause greater than 50%inhibition at 10 μM. As noted in FIG. 8, compound B showed inhibition ofgreater than 50% at a dose of 20 μM. Therefore, depending on the dosagelevel used in a single dose test, some compounds may be screened outthat otherwise may be active at slightly higher dosages. The dosage usedis subjective and may be lowered after active compounds are found atcertain levels to identify even more potent compounds.

Experimental Example 3 Apoptosis Assay

Reference compounds and test compounds were analyzed for their novel PDEinhibitory activity in accordance with the protocols for the assay,supra. In accordance with those protocols, FIG. 10 shows the effects ofsulindac sulfide and exisulind on apoptotic and necrotic cell death.HT-29 cells were treated for six days with the indicated dose of eithersulindac sulfide or exisulind. Apoptotic and necrotic cell death wasdetermined previously (Duke and Cohen, In: Current Protocols inImmunology, 3.17.1-3.17.16, New York, John Wiley and Sons, 1992). Thedata show that both sulindac sulfide and exisulind are capable ofcausing apoptotic cell death without inducing necrosis. All data werecollected from the same experiment.

FIG. 11 shows the effect of sulindac sulfide and exisulind on tumorgrowth inhibition and apoptosis induction as determined by DNAfragmentation. Top FIG. (11A); growth inhibition (open symbols, leftaxis) and DNA fragmentation (closed symbols, right axis) by exisulind.Bottom FIG. (11B); growth inhibition (open symbols) and DNAfragmentation (closed symbols) by sulindac sulfide. Growth inhibitionwas determined by the SRB assay after six days of treatment. DNAfragmentation was determined after 48 hours of treatment. All data werecollected from the same experiment.

FIG. 12 shows the apoptosis inducing properties of compound E. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof compound E for 48 hours and apoptosis was determined by the DNAfragmentation assay. The calculated EC₅₀ value was 0.05 μM.

FIG. 13 shows the apoptosis inducing properties of compound B. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof compound B for 48 hours and apoptosis was determined by the DNAfragmentation assay. The calculated EC₅₀ value was approximately 175 μM.

TABLE 4 Apoptosis-inducing activity among a series of compoundsReference compounds Fold induction at 100 μM Indomethacin <2.0 MY54454.7 Sulindac sulfide 7.9 Exisulind <2.0 E4021 <2.0 Zaprinast <2.0Sildenafil <2.0 EHNA <2.0 Test compounds Fold induction at 100 μM A <2.0B 3.4 C 5.6 D <2.0 E 4.6

In accordance with the fold induction protocol, supra, the compounds Athrough E were tested for apoptosis inducing activity, as reported inTable 4 above. Compounds B, C and E showed significant apoptoticinducing activity, greater than 2.0 fold, at a dosage of 100 μM. Ofthese three compounds, at this dosage only B and E did not inhibit COXbut did inhibit cGMP-specific PDE.

The apoptosis inducing activity for a series of phosphodiesteraseinhibitors was determined. The data are presented in Table 5 below.HT-29 cell were treated for 6 days with various inhibitors ofphosphodiesterase. Apoptosis and necrosis were determinedmorphologically after acridine orange and ethidium bromide labeling inaccordance with the assay described, supra. The data show that the novelcGMP-specific PDE is useful for screening compounds that induceapoptosis of HT-29 cells.

TABLE 5 Apoptosis-Induction Data for PDE Inhibitors Inhibitor ReportedSelectivity % Apoptosis % Necrosis Vehicle 8 6 8-methoxy-IBMX PDE1 2 1Milrinone PDE3 18 0 RO-20-1724 PDE4 11 2 MY5445 PDE5 80 5 IBMXNon-selective 4 13

Experimental Example 4 Growth Inhibition Assay

Reference compounds and test compounds were analyzed for their PDE5inhibitory activity in accordance with the protocol for the assay supra.FIG. 14 shows the inhibitory effect of various concentrations ofsulindac sulfide and exisulind on the growth of HT-29 cells. HT-29 cellswere treated for six days with various doses of exisulind (triangles) orsulindac sulfide (squares) as indicated. Cell number was measured by asulforhodamine assay as previously described (Piazza et al., CancerResearch, 55: 3110-3116, 1995). The IC₅₀ value for sulindac sulfide wasapproximately 45 μM and 200 μM for exisulind. The data show that bothsulindac sulfide and exisulind are capable of inhibiting tumor cellgrowth.

FIG. 15 shows the growth inhibitory and apoptosis-inducing activity ofsulindac sulfide. A time course experiment is shown involving HT-29cells treated with either vehicle, 0.1% DMSO (open symbols) or sulindacsulfide, 120 μM (closed symbols). Growth inhibition (15A top) wasmeasured by counting viable cells after trypan blue staining. Apoptosis(15B bottom) was measured by morphological determination followingstaining with acridine orange and ethidium bromide as describedpreviously (Duke and Cohen, in: Current Protocols in Immunology,3.17.1-3.17.16, New York, John Wiley and Sons, 1992). The datademonstrate that sulindac sulfide is capable of inhibiting tumor cellgrowth, and that the effect is accompanied by an increase in apoptosis.All data were collected from the same experiment.

FIG. 16 shows the growth inhibitory activity of test compound E. HT-29colon adenocarcinoma cells were treated with the indicated concentrationof compound E for six days and cell number was determined by the SRBassay. The calculated IC₅₀ value was 0.04 μM.

TABLE 6 Growth-inhibitory activity among a series of compounds Referencecompounds % Inhibition at 100 μM Indomethacin 75 MY5445 88 Sulindacsulfide 88 Exisulind <50 E4021 <50 sildenafil <50 zaprinast <50 Testcompounds % Inhibition at 100 μM A 68 B 77 C 80 D 78 E 62

In accordance with the screening protocol of section supra, compounds Athrough E were tested for growth inhibitory activity, as reported inTable 6 above. All the test compounds showed activity exceeding a 100 μMsingle dose test.

The growth inhibitory activity for a series of phosphodiesteraseinhibitors was determined. The data are shown in Table 7 below. HT-29cells were treated for 6 days with various inhibitors ofphosphodiesterase. Cell growth was determined by the SRB assaydescribed, supra. The data below taken with those above show thatinhibitors of the novel PDE were effective for inhibiting tumor cellgrowth.

TABLE 7 Growth Inhibitory Data for PDE Inhibitors Growth inhibitionInhibitor Reported Selectivity (IC₅₀, μM) 8-methoxy-IBMX PDE1 >200 μMMilrinone PDE3 >200 μM RO-20-1724 PDE4 >200 μM MY5445 PDE5    5 μM IBMXNon-selective >100 μM Zaprinast PDE5 >100 μM Sildenafil PDE5 >100 μME4021 PDE5 >100 μM

To show the effectiveness of this screening method on various forms ofneoplasia, compounds were tested on numerous cell lines. The effects ofsulindac sulfide and exisulind on various cell lines were determined.The data are shown in Table 8 below. The IC₅₀ values were determined bythe SRB assay. The data show the broad effectiveness of these compoundson a broad range of neoplasias, with effectiveness at comparable doserange. Therefore, compounds identified and selected by this inventionshould be useful for treating multiple forms of neoplasia.

TABLE 8 Growth Inhibitory Data of Various Cell Lines Cell Type/ IC₅₀(μM) Tissue specificity Sulindac sulfide Exisulind Compound E* HT-29,Colon 60 120 0.10 HCT116, Colon 45 90 MCF7/S, Breast 30 90 UACC375,Melanoma 50 100 A-427, Lung 90 130 Bronchial Epithelial 30 90 Cells NRK,Kidney (non ras- 50 180 transformed) KNRK, Kidney (ras 60 240transformed) Human Prostate 82 0.90 Carcinoma PC3 Colo 205 1.62 DU-1450.10 HCT-15 0.60 MDA-MB-231 0.08 MDA-MB-435 0.04 *Determined by neutralred assay as described by Schmid et al., in Proc. AACR Vol 39, p. 195(1998).

Experimental Example 5 Activity in Mammary Gland Organ Culture Model

FIG. 17 shows the inhibition of premalignant lesions in mammary glandorgan culture by sulindac metabolites. Mammary gland organ cultureexperiment were performed as previously described (Mehta and Moon,Cancer Research, 46: 5832-5835, 1986). The results demonstrate thatsulindac and exisulind effectively inhibit the formation of premalignantlesions, while sulindac sulfide was inactive.

The data support the hypothesis that cyclooxygenase inhibition is notnecessary for the anti-neoplastic properties of desired compounds.

Analysis

To select compounds for treating neoplasia, this invention provides arationale for comparing experimental data of test compounds from severalprotocols. Within the framework of this invention, test compounds can beranked according to their potential use for treating neoplasia inhumans. Those compounds having desirable effects may be selected foradditional testing and subsequent human use.

Qualitative data of various test compounds and the several protocols areshown in Table 9 below. The data show that exisulind, compound B andcompound E exhibit the appropriate activity to pass the screen of fourassays: lack of COX inhibition, and presence of effective cGMP-specificPDE inhibition, growth inhibition and apoptosis induction. The activityof these compounds in the mammary gland organ culture validates theeffectiveness of this invention. The qualitative valuations of thescreening protocols rank compound E best, then compound B and thenexisulind.

TABLE 9 Activity Profile of Various Compounds Mammary Gland COX PDEGrowth Organ Compound Inhibition Inhibition Inhibition Apoptosis CultureExisulind − + + + + + + + + + Sulindac + + + + + + + + + + + + + −sulfide MY5445 + + + + + + + + + + + + + + A − − + + + + + + + B− + + + + + + + + + + + D − − + + − − E− + + + + + + + + + + + + + + + + F − − + + + − G − − + + + + + + + + H− − + + − − Table 9 Code: Activity of compounds based on evaluating aseries of experiments involving tests for maximal activity and potency.− Not active + Slightly active + + Moderately active + + + Stronglyactive + + + + Highly active

Also disclosed is a novel assay for PKG activity, which is used in thescreening methods of this invention, but also has more generalusefulness in assaying for PKG activity for other purposes (e.g., forstudying the role of PKG in normal cellular function). For explanationpurposes, it is useful to describe the PKG assay first, beforedescribing how PKG activity can be useful in drug evaluation inascertaining whether a compound is potentially useful in the treatmentof neoplasia.

The Novel PKG Assay

The novel PKG assay of this invention involves binding to solid phaseplural amino acid sequences, each of which contain at least thecGMP-binding (cGB) domain and the phosphorylation site ofphosphodiesterase type 5 (“PDE5”). That sequence is known and describedin the literature below. Preferably, the bound PDE5 sequence does notinclude the catalytic domain of PDE5 as described below. One way to bindthe PDE5 sequences to a solid phase is to express those sequences as afusion protein of the PDE5 sequence and one member of an amino acidbinding pair, and chemically link the other member of that amino acidbinding pair to a solid phase (e.g., beads). One binding pair that canbe used is glutathione S-transferase (“GST”) and glutathione (“GSH”),with the GST being expressed as a fusion protein with the PDE5 sequencedescribed above, and the GSH bound covalently to the solid phase. Inthis fashion, the PDE5 sequence/GST fusion protein can be bound to asolid phase simply by passing a solution containing the fusion proteinover the solid phase, as described below.

RT-PCR method is used to obtain the cGB domain of PDE5 with forward andreverse primers designed from bovine PDE5A cDNA sequence(McAllister-Lucas L. M. et al, J. Biol. Chem. 268, 22863-22873, 1993)and the selection among PDE 1-10 families. 5′-3′, Inc. kits for totalRNA followed by oligo (dT) column purification of mRNA are used withHT-29 cells. Forward primer (GAA-TTC-TGT-TAG-AAA-AGC-CAC-CAG-AGA-AAT-G,203-227) and reverse primer (CTC-GAG-CTC-TCT-TGT-TTC-TTC-CTC-TGC-TG,1664-1686) are used to synthesize the 1484 bp fragment coding for thephosphorylation site and both low and high affinity cGMP binding sitesof human PDE5A (203-1686 bp, cGB-PDE5). The synthesized cGB-PDE5nucleotide fragment codes for 494 amino acids with 97% similarity tobovine PDE5A. It is then cloned into pGEX-5X-3 glutathione-S-transferase(GST) fusion vector (Pharmacia Biotech )with tac promoter, and EcoRI andXhoI cut sites. The fusion vector is then transfected into E. Coli BL21(DE3) bacteria (Invitrogen). The transfected BL21 bacteria are grown tolog phase and then IPTG is added as an inducer. The induction is carriedout at 20° C. for 24 hrs. The bacteria are harvested and lysed. Thesoluble cell lysate is incubated with GSH conjugated Sepharose 4B(GSH-Sepharose 4B). The GST-cGB-PDE5 fusion protein can bind to theGSH-Sepharose beads, and the other proteins are washed off from thebeads with excessive cold PBS.

The expressed GST-cGB-PDE5 fusion protein is displayed on 7.5% SDS-PAGEgel as an 85 Kd protein. It is characterized by its cGMP binding andphosphorylation by protein kinases G and A. It displays two cGMP bindingsites and the K_(d) is 1.6±0.2 μM, which is close to K_(d)=1.3 μM of thenative bovine PDE5. The GST-cGB-PDE5 on GSH conjugated sepharose beadscan be phosphorylated in vitro by cGMP-dependent protein kinase andcAMP-dependent protein kinase A. The K_(m) of GST-cGB-PDE5phosphorylation by PKG is 2.7 μM and the Vmax is 2.8 μM, while the K_(m)of BPDEtide phosphorylation is 68 μM. The phosphorylation by PKG showsmolecular phosphate incorporated into GST-cGB-PDE5 protein on aone-to-one ratio.

To assay a liquid sample believed to contain PKG using the PDE5-boundsolid phase described above, the sample and the solid phase are mixedwith phosphorylation buffer containing ³²P-γ-ATP. The solution isincubated for 30 minutes at 30° C. to allow for phosphorylation of thePDE5 sequence by PKG to occur, if PKG is present. The solid phase isthen separated from solution (e.g., by centrifugation or filtration) andwashed with phosphate-buffered saline (“PBS”) to remove any remainingsolution and to remove any unreacted ³²P-γ-ATP.

The solid phase can then be tested directly (e.g., by liquidscintillation counter) to ascertain whether ³²P is incorporated. If ³²Pis incorporated, that indicates that the sample contained PKG since PKGphosphorylates PDE5. If the PDE5 is bound via fusion protein, asdescribed above, the PDE5-containing fusion protein can be eluted fromthe solid phase with SDS buffer, and the eluent can be assayed for ³²Pincorporation. This is particularly advantageous if there is thepossibility that other proteins are present, since the eluent can beprocessed (e.g., by gel separation) to separate various proteins fromeach other so that the fusion protein fraction can be assayed for ³²Pincorporation. The phosphorylated fusion protein can be eluted from thesolid phase with SDS buffer and further resolved by electrophoresis. Ifgel separation is performed, the proteins can be stained to see theposition(s) of the protein, and ³²P phosphorylation of the PDE5 portionof the fusion protein by PKG can be measured by exposure of the gel toX-ray film. If ³²P is made visible on X-ray film, that indicates thatthe original sample contained PKG, which phosphorylated the PDE5 portionof the fusion protein eluted from the solid phase.

Preferably in the assay, one should add to the assay buffer an excess(e.g., 100 fold of IC₅₀ value) of protein kinase inhibitor (“PKI”) whichspecifically and potently inhibits protein kinase A (“PKA”) withoutinhibiting PKG. Inhibiting PKA is desirable since it may contribute tothe phosphorylation of the PKG substrate (e.g., PDE5). By adding PKI,any contribution to phosphorylation by PKA will be eliminated, and anyphosphorylation detected is highly likely to be due to PKG alone.

A kit can be made for the assay of this invention, which kit containsthe following pre-packaged reagents in separate containers:

1. Cell lysis buffer: 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA,1 mM Na₃VO₄, 1 mM NaF, 500 μM IBMX, proteinase inhibitors.

2. Protein kinase G solid phase substrate: recombinant GST-cGB-PDE5bound Sepharose 4B (50% slurry).

3. 2× Phosphorylation buffer: ³²P-γ-ATP (3000 mCi/mmol, 5˜10 μCi/assay),10 mM KH₂PO₄, 10 mM K₂HPO₄, 200 μM ATP, 5 mM MgCl₂.

4. PKA Protein Kinase I Inhibitor

Disposable containers and the like in which to perform the abovereactions can also be provided in the kit.

From the above, one skilled in the analytical arts will readily envisionvarious ways to adapt the assay formats described to still otherformats. In short, using at least a portion of PDE5 (or any otherprotein that can be selectively phosphorylated by PKG), the presence andrelative amount (as compared to a control) of PKG can be ascertained byevaluating phosphorylation of the phosphorylatable protein, using alabeled phosphorylation agent.

SAANDs Increase PKG Activity in Neoplastic Cells

Using the PKG assay described above, the following experiments wereperformed to establish that SAANDs increase PKG activity due either toincrease in PKG expression or an increase in cGMP levels (or both) inneoplastic cells treated with a SAAND.

Test Procedures

Two different types of PDE inhibitors were evaluated for their effectson PKG in neoplastic cells. A SAAND, exisulind, was evaluated since itis anti-neoplastic. Also, a non-SAAND classic PDE5 inhibitor, E4021, wasevaluated to ascertain whether PKG elevation was simply due to classicPDE5 inhibition, or whether PKG elevation was involved in thepro-apoptotic effect of SAANDs inhibition of PDE5 and the novel PDEdisclosed in U.S. patent application Ser. No. 09/173,375 to Liu et alfiled Oct. 15, 1998.

To test the effect of cGMP-specific PDE inhibition on neoplasiacontaining the APC mutation, SW480 colon cancer cells were employed. SW480 is known to contain the APC mutation. About 5 million SW480 cells inRPMI 5% serum are added to each of 8 dishes:

2-10 cm dishes—30 μL DMSO vehicle control (without drug),

3-10 cm dishes—200 μM, 400 μM, 600 μM exisulind, and

3-10 cm dishes—E4021; 0.1 μM, 1 μM and 10 μM.

The dishes are incubated for 48 hrs at 37° C. in 5% CO₂ incubator.

The liquid media are aspirated from the dishes (the cells will attachthemselves to the dishes). The attached cells are washed in each dishwith cold PBS, and 200 μL cell lysis buffer (i.e., 50 mM Tris-HCl, 1%NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 500 μM IBMX withproteinase inhibitors) is added to each dish. Immediately after the celllysis buffer is added, the lysed cells are collected by scraping thecells off each dish. The cell lysate from each dish is transferred to amicrofuge tube, and the microfuge tubes are incubated at 4° C. for 15minutes while gently agitating the microfuge tubes to allow the cells tolyse completely. After lysis is complete, the microfuge tubes arecentrifuged full speed (14,000 r.p.m.) for 15 minutes. The supernatantfrom each microfuge tube is transferred to a fresh microfuge tube.

A protein assay is then performed on the contents of each microfuge tubebecause the amount of total protein will be greater in the control thanin the drug-treated samples, if the drug inhibits cell growth.Obviously, if the drug does not work, the total protein in thedrug-treated samples should be virtually the same as control. In theabove situation, the control and the E-4021 microfuge tubes neededdilution to normalize them to the high-dose exisulind-treated samples(the lower dose groups of exisulind had to be normalized to the highestdose exisulind sample). Thus, after the protein assays are performed,the total protein concentration of the various samples must benormalized (e.g., by dilution).

For each drug concentration and control, two PKG assays are performed,one with added cGMP, and one without added cGMP, as described in detailbelow. The reason for performing these two different PKG assays is thatcGMP specifically activates PKG. When PKG activity is assayed using thenovel PKG assay of this invention, one cannot ascertain whether anyincrease the PKG activity is due to increased cGMP in the cells (thatmay be caused by cGMP-specific PDE inhibition) or whether the PKGactivity level is due to an increased expression of PKG protein. Bydetermining PKG activity in the same sample both with and without addedcGMP, one can ascertain whether the PKG activity increase, if any, isdue to increased PKG expression. Thus, if an anti-neoplastic drugelevates PKG activity relative to control, one can establish if thedrug-induced increase is due to increased PKG protein expression (asopposed to activation) in the drug-treated sample if (1) thedrug-treated sample with extra cGMP exhibits greater PKG activitycompared to the control sample with extra cGMP, and (2) the drug-treatedsample without extra cGMP exhibits greater PKG activity relative tocontrol.

After parallel samples with and without added cGMP are prepared, 50 μLof each cell lysate is added to 20 μL of the PDE5/GST solid phasesubstrate slurry described above. For each control or drug cell lysatesample to be evaluated, the reaction is started by addingphosphorylation buffer containing 10 μCi ³²P-γ-ATP solution (200 μM ATP,4.5 mM MgCl; 5 mM KH₂PO₄; 5 mM K₂HPO₄;) to each mixture. The resultantmixtures are incubated at 30° C. for 30 minutes. The mixtures are thencentrifuged to separate the solid phase, and the supernatant isdiscarded. The solid phase in each tube is washed with 700 μL cold PBS.To the solid phase, Laemmli sample buffer (Bio-Rad) (30 μL) is added.The mixtures are boiled for 5 minutes, and loaded onto 7.5% SDS-PAGE.The gel is run at 150 V for one hour. The bands obtained are stainedwith commassie blue to visualize the 85 Kd GST-PDE5 fusion proteinbands, if present. The gel is dried, and the gel is laid on x-ray filmwhich, if the PDE5 is phosphorylated, the film will show a correspondingdarkened band. The darkness of each band relates to the degree ofphosphorylation.

As shown in FIGS. 18A and 18B, the SAAND exisulind causes PKG activityto increase in a dose-dependent manner in both the samples with addedcGMP and without added cGMP relative to the control samples with andwithout extra cGMP. This is evidenced by the darker appearances of the85 Kd bands in each of the drug-treated samples. In addition, the SW480samples treated with exisulind show a greater PKG phosphorylationactivity with added cGMP in the assay relative to the samples treatedwith vehicle with added cGMP. Thus, the increase in PKG activity in thedrug-treated samples is not due only to the activation of PKG by theincrease in cellular cGMP when the SAAND inhibits cGMP-specific PDE, theincrease in PKG activity in neoplasia harboring the APC mutation is dueto increased PKG expression as well.

Also the fact that the E4021-treated SW480 samples do not exhibit PKGactivation relative to control (see FIGS. 18A and 18B) shows that theincreased PKG activation caused by SAANDs in neoplasia containing theAPC mutation is not simply due to inhibition of classic PDE5.

As an analytic technique for evaluating PKG activation, instead of x-rayfilm exposure as described above, the 85 Kd band from the SDS page canbe evaluated for the degree of phosphorylation by cutting the band fromthe gel, and any ³²P incorporated in the removed band can be counted byscintillation (beta) counter in the ³²P window.

To test the effect of cGMP-specific PDE inhibition on neoplasiacontaining the β-catenin mutation, HCT116 colon cancer cells wereemployed. HCT116 is known to contain the β-catenin mutation, but isknown not to contain the APC mutation.

The same procedure is used to grow the HCT116 cells as is used in theSW480 procedure described above. In this experiment, only exisulind andcontrols were used. The exisulind-treated cells yielded PKG that wasphosphorylated to a greater extent than the corresponding controls,indicating that PKG activation occurred in the drug-treated cells thatis independent of the APC mutation.

Thus, for the purposes of the present invention, “β-catenin” refers towild type and/or mutant forms of that protein.

Confirmation of Increased PKG Expression and Decreased β-Catenin in SW480 by Western Blot

As demonstrated above, SAANDs cause an increase in PKG expression and anincrease in cGMP level, both of which cause an increase in PKG activityin SAANDs-treated neoplastic cells. This increase in PKG proteinexpression was further verified by relatively quantitative western blot,as described below.

SW480 cells treated with exisulind as described previously are harvestedfrom the microfuge tubes by rinsing once with ice-cold PBS. The cellsare lysed by modified RIPA buffer for 15 minutes with agitation. Thecell lysate is spun down in a cold room. The supernatants aretransferred to fresh microcentrifuge tubes immediately after spinning.BioRad DC Protein Assay (Temecula, Calif.) is performed to determine theprotein concentrations in samples. The samples are normalized forprotein concentration, as described above.

50 μg of each sample is loaded onto a 10% SDS gel. SDS-PAGE isperformed, and the proteins then are transferred to a nitrocellulosemembrane. The blotted nitrocellulose membrane is blocked in freshlyprepared TBST containing 5% nonfat dry milk for one hour at roomtemperature with constant agitation.

A goat-anti-PKG primary antibody is diluted to the recommendedconcentration/dilution in fresh TBST/5% nonfat dry milk. Thenitrocellulose membrane is placed in the primary antibody solution andincubated one hour at room temperature with agitation. Thenitrocellulose membrane is washed three times for ten minutes each withTBST. The nitrocellulose membrane is incubated in a solution containinga secondary peroxidase (POD) conjugated rabbit anti-goat antibody for 1hour at room temperature with agitation. The nitrocellulose membrane iswashed three times for ten minutes each time with TBST. The detection isperformed by using Boehringer Mannheim BM blue POD substrate.

As graphically illustrated in FIG. 19, exisulind causes the drop ofβ-catenin and the increase of PKG, which data were obtained by Westernblot. SW480 cells were treated with exisulind or vehicle (0.1% DMSO) for48 hours. 50 μg supernatant of each cell lysate was loaded onto a 10%SDS-gel and blotted to a nitrocellulose membrane, and the membrane wasprobed with rabbit-anti-β-catenin and rabbit anti-PKG antibodies.

SAANDs Reduce β-Catenin Levels in Neoplastic Cells

This observation was made by culturing SW480 cells with either 200, 400or 600 μM exisulind or vehicle (0.1% DMSO). The cells are harvested 48hours post treatment and processed for immunoblotting. Immuno-reactiveprotein can be detected by Western blot. Western blot analysisdemonstrated that expression of β-catenin was reduced by 50% in theexisulind-treated cells as compared to control. These results indicatethat β-catenin is reduced by SAANDs treatment. Together with the resultsabove, establishing PKG activity increases with such treatment, and theresults below, establishing that β-catenin is phosphorylated by PKG,these results indicate that the reduction of β-catenin in neoplasticcells is initiated by activation of PKG. Thus, using PKG activity inneoplasia as a screening tool to select compounds as anti-neoplastics isuseful.

The Phosphorylation of β-catenin by PKG

In vitro PKG phosphorylates β-catenin. The experiment that establishedthis involves immunoprecipitating the β-catenin-containing complex fromSW480 cells (not treated with any drug) in the manner described belowunder “β-catenin immunoprecipitation.” The immunoprecipitated complex,while still trapped on the solid phase (i.e., beads) is mixed with³²P-γ-ATP and pure PKG (100 units). Corresponding controls without addedPKG are prepared.

The protein is released from the solid phase by SDS buffer, and theprotein-containing mixture is run on a 7.5% SDS-PAGE gel. The running ofthe mixture on the gel removes excess ³²P-γ-ATP from the mixture. Any³²P-γ-ATP detected in the 93Kd β-catenin band, therefore, is due to thephosphorylation of the β-catenin. Any increase in ³²P-γ-ATP detected inthe 93 Kd β-catenin band treated with extra PKG relative to the controlwithout extra PKG, is due to the phosphorylation of the β-catenin in thetreated band by the extra PKG.

The results we obtained were that there was a noticeable increase inphosphorylation in the band treated with PKG as compared to the control,which exhibited minimal, virtually undetectable phosphorylation. Thisresult indicates that β-catenin can be phosphorylated by PKG.

The Phosphorylation of Mutant β-Catenin by PKG

The same procedure described in the immediately preceding section wasperformed with HCT116 cells, which contain no APC mutation, but containa β-catenin mutation. The results of those experiments also indicatethat mutant β-catenin is phosphorylated by PKG.

Thus, for the purposes of the present invention, the phosphorylation ofβ-catenin refers to the phosphorylation of wild type and/or mutant formsof that protein.

β-Catenin Precipitates with PKG

Supernatants of both SW480 and HCT116 cell lysates are prepared in thesame way described above in the Western Blot experiments. The celllysates are pre-cleared by adding 150 μl of protein A Sepharose beadslurry (50%) per 500 μg of cell lysate and incubating at 4° C. for 10minutes on a tube shaker. The protein A beads are removed bycentrifugation at 14,000×g at 4° C. for 10 minutes. The supernatants aretransferred to a fresh centrifuge tubes. 10 μg of the rabbit polyclonalanti-β-catenin antibody (Upstate Biotechnology, Lake Placid, N.Y.) areadded to 500 μg of cell lysate. The cell lysate/antibody mixture isgently mixed for 2 hours at 4° C. on a tube shaker. The immunocomplex iscaptured by adding 150 μl protein A Sepharose bead slurry (75 μl packedbeads) and by gently rocking the mixture on a tube shaker for overnightat 4° C. The Sepharose beads are collected by pulse centrifugation (5seconds in the microcentrifuge at 14,000 rpm). The supernatant fractionis discarded, and the beads are washed 3 times with 800 μl ice-cold PBSbuffer. The Sepharose beads are resuspended in 150 μl 2× sample bufferand mixed gently. The Sepharose beads are boiled for 5 minutes todissociate the immunocomplexes from the beads. The beads are collectedby centrifugation and SDS-PAGE is performed on the supernatant.

A Western blot is run on the supernatant, and the membrane is thenprobed with a rabbit-anti-β-catenin antibody. Then the membrane iswashed 3 times for 10 minutes each time with TBST to remove excessanti-β-catenin antibody. A goat, anti-rabbit antibody conjugated tohorseradish peroxidase is added, followed by a one hour incubation atroom temperature. When that is done, one can visualize the presence ofβ-catenin with an HRPO substrate. In this experiment, we could clearlyvisualize the presence of β-catenin.

To detect PKG on the same membrane, the anti-β-catenin antibodyconjugate is first stripped from the membrane with a 62 mM tris-HClbuffer (pH 7.6) with 2% SDS and 100 μM 2β-mercaptoethanol in 55° C.water bath for 0.5 hour. The stripped membrane is then blocked in TBSTwith 5% non-fat dried milk for one hour at room temperature whileagitating the membrane. The blocked, stripped membrane is then probedwith rabbit polyclonal anti-PKG antibody (Calbiochem, LaJolla, Calif.),that is detected with goat, anti-rabbit second antibody conjugated toHRPO. The presence of PKG on the blot membrane is visualized with anHRPO substrate. In this experiment, the PKG was, in fact, visualized.Given that the only proteins on the membrane are those thatimmunoprecipitated with β-catenin in the cell supernatants, this resultclearly establishes that PKG was physically linked to the proteincomplex containing the β-catenin in the cell supernatants.

The same Western blot membrane was also probed after stripping withanti-GSK3-β antibody to ascertain whether it also co-precipitated withβ-catenin. In that experiment, we also detected GSK3-β on the membrane,indicating that the GSK3-β precipitated with the GSK3-β and PKG,suggesting that the three proteins may be part of the same complex.Since GSK3-β and β-catenin form part of the APC complex in normal cells,this indicates that PKG may be part of the same complex, and may beinvolved in the phosphorylation of β-catenin as part of that complex.

Anti-Neoplastic Pharmaceutical Compositions Containing cGMP PDEInhibitors

One drug that was also invented before its mechanism of action was foundto involve cGMP inhibition and before it was known to meet the selectioncriterion of this invention is(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride (“Compound I”) (see “General Schemes for ProducingcGMP-Specific PDE Inhibitors” above). It has been demonstrated in invitro and in vivo evaluations as anti-neoplastic having activitiesagainst a broad range of neoplasias. It is also safe in animal studiesand in a single, escalating dose human study.

As one skilled in the art will recognize from the data presented below,Compound I can safely be given to animals at doses far beyond thetolerable (and in many cases toxic) doses of conventionalchemotherapeutics or anti-neoplastic NSAIDs. For example, in an acutetoxicity study in rats, single oral doses of Compound I administered (ina 0.5% carboxy-methylcellulose vehicle) at doses up to and including2000 mg/kg resulted in no observable signs of toxicity. At 4000 mg/kg,body weight gains were slightly reduced. A single dose of 1000 mg/kgadministered intraperitoneally resulted in reduced body weight gain,with mesenteric adhesions seen in some animals from this group atnecropsy.

In dogs, the administration of Compound I in capsules at 1000 mg/kgresulted in no signs of toxicity to the single group of two male and twofemale dogs. Due to the nature of Compound I capsules, this dosenecessitated the use of at least 13 capsules to each animal, which wasjudged to be the maximum number without subjecting the animals tostress. Therefore, these dogs were subsequently administered sevenconsecutive doses of 1000 mg/kg/day. At no time in either dosing phasewere any obvious signs of drug-related effects observed.

Thus, on a single-dose basis, Compound I is not acutely toxic. Based onthe findings of these studies, the oral LD₅₀ of Compound I wasconsidered to be greater than 1000 mg/kg in dogs and 4000 mg/kg in rats,and the intraperitoneal LD₅₀ was considered to be greater than 1000mg/kg in rats.

A seven-day dose-range finding study in rats, where Compound I wasevaluated by administering it at doses of 0, 50, 500 or 2000 mg/kg/dayresulted in no observable signs of toxicity at 50 mg/kg/day. At 500mg/kg/day, treatment-related effects were limited to an increase inabsolute and relative liver weights in female rats. At 2000 mg/kg/day,effects included labored breathing and/or abnormal respiratory sounds,decreased weights gains and food consumption in male rats, and increasedliver weights in female rats. No hematological or blood chemistrychanges nor any microscopic pathology changes, were seen at any doselevel.

A 28-day study in rats was also carried out at 0, 50, 500 and 2000mg/kg/day. There were no abnormal clinical observations attributed toCompound I, and body weight changes, ophthalmoscopic examinations,hematological and blood chemistry values and urinalysis examinationswere unremarkable. No macroscopic tissue changes were seen at necropsy.Organ weight data revealed statistically significant increase in liverweights at 2000 mg/kg/day, and statistically significant increases inthyroid weights for the 2000 mg/kg/day group. The slight increases atthe lower doses were not statistically significant. Histopathologicalevaluation of tissues indicated the presence of traces of follicularcell hypertrophy, increased numbers of mitotic figures (suggestive ofpossible cell proliferation) in the thyroid gland and mild centrilobularhypertrophy in the liver. These changes were generally limited to asmall number of animals at the 2000 mg/kg/day dose, although one femaleat 500 mg/kg/day had increased mitotic figures in the thyroid gland. Thefindings in the liver may be indicative of a very mild stimulation ofmicrosomal enzymes, resulting in increased metabolism of thyroidhormones, which in turn resulted in thyroid stimulation. Thus, oneskilled in the art will recognize that these effects are extremelyminimal compared to what one would expect at similar doses ofconventional chemotherapeutics or NSAIDs.

To further establish the safety profile of Compound I, a study wasperformed to evaluate whether Compound I-induced apoptosis of prostatetumor cell lines was comparable to its effects on prostate epithelialcells derived from normal tissue. The androgen-sensitive prostate tumorcell line, LNCaP (from ATCC (Rockville, Md.)) was propagated understandard conditions using RPMI 160 medium containing 5% fetal calveserum and 2 mM glutamine. Primary prostate epithelial cell cultures(PrEC) derived from normal prostate (from Clonetics Inc. (San Diego,Calif.)) were grown under the same conditions as the tumor cell lineexcept a serum-free medium optimized for the growth of such cultures wasused (Clonetics Inc). For the experiments, LNCaP or PrEC cells wereseeded in 96 well plates at a density of 10,000 cells per well. After 24hours, the cells were treated with either vehicle (0.1% DMSO) or 50 μMCompound I (free base) solubilized in DMSO. After various drug treatmenttimes (4, 24, 48, 72, or 99 hours) the cells were lysed and processedfor measurement of histone-associated DNA as an indicator of apoptoticcell death (see, Piazza et al., Cancer Research 57: 2452-2459, 1997).

FIG. 27 shows a time-dependent increase in the amount ofhistone-associated fragmented DNA in LNCaP cell cultures followingtreatment with 50 μM Compound I(free base). A significant increase infragmented DNA was detected after 24 hours of treatment, and theinduction was sustained for up to 4 days of continuous treatment. Bycontrast, treatment of PrEC (“normal” prostate) cells with Compound I(50 μM) did not affect DNA fragmentation for up to 4 days of treatment.These results demonstrate a selective induction of apoptosis inneoplastic cells, as opposed to normal cells. This is in marked contrastto conventional chemotherapeutics that induce apoptosis or necrosis inrapidly growing normal and neoplastic cells alike.

Identification of Additional Inhibitors

As to identifying structurally additional cGMP-specific PDE inhibitingcompounds that can be effective therapeutically as anti-neoplastics, oneskilled in the art has a number of useful model compounds disclosedherein (as well as their analogs incorporated by reference) that can beused as the bases for computer modeling of additional compounds havingthe same conformations but different chemically. For example, softwaresuch as that sold by Molecular Simulations Inc. release of WebLab®ViewerPro™ includes molecular visualization and chemical communicationcapabilities. Such software includes functionality, including 3Dvisualization of known active compounds to validate sketched or importedchemical structures for accuracy. In addition, the software allowsstructures to be superimposed based on user-defined features, and theuser can measure distances, angles, or dihedrals.

In this situation, since the structures of other active compounds aredisclosed above, one can apply cluster analysis and 2D and 3D similaritysearch techniques with such software to identify potential newadditional compounds that can then be screened and selected according tothe selection criteria of this invention. These software methods relyupon the principle that compounds, which look alike or have similarproperties, are more likely to have similar activity, which can beconfirmed using the selection criterion of this invention.

Likewise, when such additional compounds are computer modeled, many suchcompounds and variants thereof can be synthesized using knowncombinatorial chemistry techniques that are commonly used by those ofordinary skill in the pharmaceutical industry. Examples of a fewfor-hire combinatorial chemistry services include those offered by NewChemical Entities, Inc. of Bothell Wash., Protogene Laboratories, inc.,of Palo Alto, Calif., Axys, Inc. of South San Francisco, Calif.,Nanosyn, Inc. of Tucson, Ariz., Trega, Inc. of San Diego, Calif., andRBI, Inc. of Natick, Mass. There are a number of other for-hirecompanies. A number of large pharmaceutical companies have similar, ifnot superior, in-house capabilities. In short, one skilled in the artcan readily produce many compounds for screening from which to selectpromising compounds for treatment of neoplasia having the attributes ofcompounds disclosed herein.

In addition, there are a number of commercially-known libraries ofcompounds usually made with combinatorial techniques. Such compounds canfirst be assessed using the types of software explained above toascertain whether they are conformationally similar to active compoundsof the types disclosed herein. After identifying such conformationallysimilar compounds, the compounds can readily be screened according tothe methods of this invention to yield anti-neoplastic cGMP PDEinhibitors.

To further assist in identifying compounds that can be screened and thenselected using the criterion of this invention, knowing the binding ofselected anti-neoplastic compounds to PDE5 protein is of interest. Bythe procedures discussed below, it was found that preferable, desirablecompounds meeting the selection criteria of this invention bind to thecGMP catalytic region of PDE5.

To establish this, a PDE5 sequence that does not include the catalyticdomain was used. One way to produce such a sequence is to express thatsequence as a fusion protein, preferably with glutathione S-transferase(“GST”). Production of a GST-cGB-PDE5 fusion protein was carried out bythe procedure described above in the section entitled The Novel PKGAssay.

A cGMP binding assay for compounds of interest (Francis S. H. et al, J.Biol. Chem. 255, 620-626, 1980) is done in a total volume of 100 μLcontaining 5 mM sodium phosphate buffer (pH=6.8), 1 mM EDTA, 0.25 mg/mLBSA, H³-cGMP (2 μM, NEN) and the GST-cGB-PDE5 fusion protein (30μg/assay). Each compound to be tested is added at the same time as³H-cGMP substrate, and the mixture is incubated at 22° C. for 1 hour.Then, the mixture is transferred to Brandel MB-24 cell harvester withGF/B as the filter membrane followed by 2 washes with 10 mL of cold 5 mMpotassium buffer (pH 6.8). The membranes are then cut out andtransferred to scintillation vials followed by the addition of 1 mL ofH₂O and 6 mL of Ready Safe™ liquid scintillation cocktail to each vial.The vials are counted on a Beckman LS 6500 scintillation counter.

For calculation, blank samples are prepared by boiling the bindingprotein for 5 minutes, and the binding counts are <1% when compared tounboiled protein. The quenching by filter membrane or other debris arealso calibrated.

PDE5 inhibitors, sulfide, exisulind, Compound B, Compound E, E4021 andzaprinast, and cyclic nucleotide analogs, cAMP, cyclic IMP,8-bromo-cGMP, cyclic UMP, cyclic CMP, 8-bromo-cAMP, 2′-O-butyl-cGMP and2′-O-butyl-cAMP are selected to test whether they could competitivelybind to the cGMP binding sites of the GST-cGB-PDE5 protein. The resultswere shown in FIG. 24. cGMP specifically binds GST-cGB-PDE5 protein.Cyclic AMP, cUMP, cCMP, 8-bromo-cAMP, 2′-O-butyl-cAMP and2′-O-butyl-cGMP did not compete with cGMP in binding. Cyclic IMP and8-bromo-cGMP at high concentration (100 μM) can partially compete withcGMP (2 μM) binding. None of the PDE5 inhibitors showed any competitionwith cGMP in binding of GST-cGB-PDE5. Therefore, they do not bind to thecGMP binding sites of PDE5.

However, Compound E does competitively (with cGMP) bind to PDE 5 (i.e.,Peak A). (Compound E also competitively (with cGMP) binds to PDE PeakB.). Given that Compound E does not bind to the cGMP-binding site ofPDE5, the fact that there is competitive binding between Compound E andcGMP at all means that desirable compounds such as Compound E bind tothe cGMP catalytic site on PDE5, information that is readily obtainableby one skilled in the art (with conventional competitive bindingexperiments) but which can assist one skilled in the art more readily tomodel other compounds. Thus, with the chemical structures of desirablecompounds presented herein and the cGMP binding site information, oneskilled in the art can model, identify and select (using the selectioncriteria of this invention) other chemical compounds for use astherapeutics.

A Human Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

The present invention discloses using a human epidermal growth tyrosinekinase inhibitor in combination with a cGMP-specific PDE inhibitor totreat a patient with cancer. One such human epidermal growth tyrosinekinase inhibitor disclosed for use herein in combination with acGMP-specific PDE inhibitor is Iressa, also called ZD1839. Iressa is aninhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK),with specificity against the EGFR.

Iressa is an orally active inhibitor which blocks signal transductionpathways implicated in promoting cancer growth (WO02/28409; WO020020;WO02/005791; WO02/002534; WO01/076586; each of which are incorporatedherein by reference). Iressa reportedly has antiangiogenic activity, ithas antitumor activity against such cancers as colon, breast, ovarian,gastric, non-small lung cancer, pancreatic prostate, and leukemia, iteliminates EGFR, HER2, and HER3 phosphorylation, it inhibits humanbreast xenograft growth and it has been used in patients (Ciardello etal., 2001, Cancer Res. 7:5:1459-1465; Moulder et al., 2001, Cancer Res.61:24:8887-8895; Barker et al., 2001, Bioorganic and Medicinal ChemistryLetters 11:14:1911-1914; Moasser et al. 2001, Cancer Res.61:19:7184-7188; Chan et al., 2002, Cancer Res. 62:1:122-128; Ranson etal., 2002, J. Clin. Oncology 20:9:2240-2250; WO01/076586).

Iressa is a quinazoline and has the chemical name 4-quinazolinamine,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-(9CI)and the chemical formula C22H24ClFN4O3. Iressa's structure is asfollows:

Combination Treatment with a Human Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitor and a cGMP-Specific PDE Inhibitor

The invention discloses a method which encompasses treating a patientwith neoplasia with both an inhibitor of human epidermal growth factorreceptor tyrosine kinase such as Iressa, and a cGMP-specific PDEinhibitor. By treating a patient with this combination, therapeuticresults can be achieved that are not seen with either therapy alone.Such combination therapy enhances the benefit to the patient withoutincreasing harmful side effects. For example, exisulind is onecGMP-specific PDE inhibitor that can be used in combination with aninhibitor of human epidermal growth factor receptor tyrosine kinase inthis invention. The invention includes Iressa, a member of the family ofinhibitors of human epidermal growth factor receptor tyrosine kinase.

As explained above, exisulind is one example of an appropriatecGMP-specific PDE inhibitor to be used in combination with an inhibitorof human epidermal growth factor receptor tyrosine kinase in thepractice of this invention. Exisulind inhibits both PDE5 and PDE2, andtreatment of neoplastic cells with exisulind results in growthinhibition and apoptosis (See Table 8). In another aspect of theinvention the cGMP-specific PDE inhibitor is(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride, also called CP461 (see “General Schemes for ProducingcGMP-Specific PDE Inhibitors,” and also see “Anti-NeoplasticPharmaceutical Compositions Containing cGMP PDE Inhibitors,” above).

Exisulind has no clinically significant side effects when administeredat its recommended dose of 300-400 mg/day. When administered at doseshigher than the recommended therapeutic levels, treatment with exisulindcan lead to elevated levels of liver enzymes. This effect is reversible,and liver enzymes return to normal levels when the administered dose ofexisulind returns to the traditionally recommended level or whentreatment is discontinued. The purpose of using combinations is to allowa lower dose of each to be used. Similarly,(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride has minimal effects on the liver (see “Anti-NeoplasticPharmaceutical Compositions Containing cGMP PDE Inhibitors,” above).

For treatment of cancer, a common dose of an inhibitor of humanepidermal growth factor receptor tyrosine kinase such as Iressa may be10-200 mg/kg/day (Chan et al., 2002, Cancer Res. 62:1:122-128; Ranson etal., 2002, J. Clin. Oncology 20:9:2240-2250). In each of theaforementioned methodologies, the inhibitor of the human epidermalgrowth factor receptor tyrosinase kinase and the PDE inhibitor may beadministered simultaneously or in succession/alternatingly. Either maybe delivered first. In one aspect of the invention the inhibitor of thehuman epidermal growth factor receptor tyrosinase kinase is Iressa. Inanother aspect of the invention the cGMP-specific PDE inhibitor isexisulind, and in yet another aspect it is(Z)-5-fluoro-2-methyl-(4-Pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride. The use of(Z)-5-fluoro-2-methyl-(4-Pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride is described more fully above in “Anti-NeoplasticPharmaceutical Compositions Containing cGMP PDE Inhibitors. Theinvention should be construed to include other human epidermal growthfactor tyrosine kinase inhibitors and cGMP-specific PDE inhibitors asdescribed herein or as known to those of skill in the art.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A method of inhibiting the growth of neoplastic lesions in a patientcomprising administering to said patient Iressa and the cGMP-specificphosphodiesterase inhibitor(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride.
 2. The method of claim 1, wherein Iressa is administeredsimultaneously with(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride.
 3. The method of claim 1, wherein Iressa is administeredalternatingly with(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride.